“
“Hippocalcin is a Ca2+-binding protein that belongs to a family of neuronal Ca2+sensors and is a key mediator of many cellular functions including synaptic plasticity and learning. However, the molecular mechanisms involved in hippocalcin signalling remain illusive. Here we studied whether glutamate receptor activation induced by locally applied or synaptically

released glutamate can be decoded by hippocalcin translocation. Local AMPA find more receptor activation resulted in fast hippocalcin-YFP translocation to specific sites within a dendritic tree mainly due to AMPA receptor-dependent depolarization and following Ca2+influx via voltage-operated calcium channels. Short local NMDA receptor activation induced fast hippocalcin-YFP translocation in a dendritic shaft at the application site due to direct Ca2+influx via NMDA receptor channels. Intrinsic network bursting produced hippocalcin-YFP translocation to a set of dendritic spines when they were subjected to several successive synaptic vesicle releases during a given burst whereas no translocation to spines was observed selleck chemicals llc in response to a single synaptic vesicle release and to back-propagating action potentials. The translocation to spines required Ca2+influx via synaptic NMDA receptors in which Mg2+ block is relieved by postsynaptic depolarization. This synaptic translocation was restricted to spine

heads and even closely (within 1–2 μm) located spines on the same dendritic branch signalled independently. Thus, we conclude that

hippocalcin may differentially decode various spatiotemporal patterns of glutamate receptor activation into site- and time-specific translocation to its targets. Hippocalcin also possesses an ability to produce local signalling at the single synaptic level providing a molecular mechanism for homosynaptic plasticity. “
“In light of anatomical evidence suggesting differential connection patterns in central vs. peripheral representations of cortical areas, we investigated the extent to which the response properties of cells in the primary visual area (V1) of the marmoset 4-Aminobutyrate aminotransferase change as a function of eccentricity. Responses to combinations of the spatial and temporal frequencies of visual stimuli were quantified for neurons with receptive fields ranging from 3° to 70° eccentricity. Optimal spatial frequencies and stimulus speeds reflected the expectation that the responses of cells throughout V1 are essentially uniform, once scaled according to the cortical magnification factor. In addition, temporal frequency tuning was similar throughout V1. However, spatial frequency tuning curves depended both on the cell’s optimal spatial frequency and on the receptive field eccentricity: cells with peripheral receptive fields showed narrower bandwidths than cells with central receptive fields that were sensitive to the same optimal spatial frequency.

“
“Hippocalcin is a Ca2+-binding protein that belongs to a family of neuronal Ca2+sensors and is a key mediator of many cellular functions including synaptic plasticity and learning. However, the molecular mechanisms involved in hippocalcin signalling remain illusive. Here we studied whether glutamate receptor activation induced by locally applied or synaptically

released glutamate can be decoded by hippocalcin translocation. Local AMPA Selleck Ku-0059436 receptor activation resulted in fast hippocalcin-YFP translocation to specific sites within a dendritic tree mainly due to AMPA receptor-dependent depolarization and following Ca2+influx via voltage-operated calcium channels. Short local NMDA receptor activation induced fast hippocalcin-YFP translocation in a dendritic shaft at the application site due to direct Ca2+influx via NMDA receptor channels. Intrinsic network bursting produced hippocalcin-YFP translocation to a set of dendritic spines when they were subjected to several successive synaptic vesicle releases during a given burst whereas no translocation to spines was observed selleck inhibitor in response to a single synaptic vesicle release and to back-propagating action potentials. The translocation to spines required Ca2+influx via synaptic NMDA receptors in which Mg2+ block is relieved by postsynaptic depolarization. This synaptic translocation was restricted to spine

heads and even closely (within 1–2 μm) located spines on the same dendritic branch signalled independently. Thus, we conclude that

hippocalcin may differentially decode various spatiotemporal patterns of glutamate receptor activation into site- and time-specific translocation to its targets. Hippocalcin also possesses an ability to produce local signalling at the single synaptic level providing a molecular mechanism for homosynaptic plasticity. “
“In light of anatomical evidence suggesting differential connection patterns in central vs. peripheral representations of cortical areas, we investigated the extent to which the response properties of cells in the primary visual area (V1) of the marmoset Megestrol Acetate change as a function of eccentricity. Responses to combinations of the spatial and temporal frequencies of visual stimuli were quantified for neurons with receptive fields ranging from 3° to 70° eccentricity. Optimal spatial frequencies and stimulus speeds reflected the expectation that the responses of cells throughout V1 are essentially uniform, once scaled according to the cortical magnification factor. In addition, temporal frequency tuning was similar throughout V1. However, spatial frequency tuning curves depended both on the cell’s optimal spatial frequency and on the receptive field eccentricity: cells with peripheral receptive fields showed narrower bandwidths than cells with central receptive fields that were sensitive to the same optimal spatial frequency.

Lawson and colleagues reported that based on 3 years of data captured by the Quarantine Activity and Reporting System (QARS), vaccine-preventable and tropical diseases are not major causes of death in international travelers Dasatinib arriving in the United States.[4] Because malaria is not a communicable disease spread person-to-person, reports of malaria are not requested by CDC Quarantine Stations. Only deaths that occurred during travel (on a conveyance or at a US port of entry) are requested. Thus, QARS did not capture 12 malaria deaths associated with international travel

reported by the US National Malaria Surveillance System during that same time period.[2] While QARS is capable of collecting travel-related illnesses or deaths, it would not be an effective surveillance system for travel-associated mortality due to malaria. The cause of death for travelers who died during travel or upon returning from travel might be captured on the US Standard Certificate of Death.[8] However, only the travel-associated data recorded on the death certificate relate to fatal travel-related injury. As a result, data on returning travelers who

died as a result of travel-related illness will not be captured systematically by the current version of the US death certificate for inclusion in UK-371804 ic50 US vital statistics data. The risks related to travel may not even be considered in assigning cause of death, especially if the signs and symptoms of disease were not overtly suggestive of

a specific travel-related illness, such as malaria or rickettsia, whose symptoms may be shared with many other less exotic maladies. While travel-related information is obtained from ill patients who are able to provide it, the value PtdIns(3,4)P2 of a travel history collected by a physician is often limited to its use in diagnosis and treatment. Travel histories collected in a clinical setting for treatment are often not collected at all or are incomplete,[9] which can limit a systematic collection of epidemiologic data related to severe travel-related illnesses. Furthermore, if the patient dies during hospitalization or while seeking treatment, an autopsy may not necessarily be performed, and thus the true cause of death remains a mystery. Autopsy rates in the United States have been steadily declining since the 1970s, with 50% of autopsies now performed on persons whose death was related to an external cause, such as assault, suicide, and accidental poisoning.[10] If a returning traveler (who truly had severe malaria) presented to an emergency department 2 weeks after returning from travel, a diagnosis of renal failure might be made based on creatinine levels.

Although erm(B) gene mediates high-level resistance and mef(A) gene correlates with low-level resistance, the rate of erythromycin-resistant S. pneumoniae isolates containing both genes is growing worldwide (Song et al., 2004a, b; Farrell et al., 2005). As the single presence of erm(B) gene determines a high macrolide resistance level,

the dual presence of erm(B) and mef(A) genes may not be advantageous in terms of bacterial survival. Thus, we postulated that pneumococcal isolates with both erm(B) and mef(A) genes originated from strains with only mef(A) gene in which the erm(B) gene was introduced; this has been supported by multilocus sequence typing (MLST) analysis (Ko & Song, 2004). However, the characteristics of pneumococcal isolates containing both erm(B) and mef(A) genes have not been investigated. check details Several investigators have reported that S. pneumoniae isolates with both erm(B) and mef(A) gene show resistance against more antimicrobial agents (Farrell DAPT supplier et al., 2004; Jenkins et al., 2008). As multidrug resistance (MDR) is linked to an increased risk of treatment failure, increased prevalence of S. pneumoniae isolates containing both erm(B) and mef(A) genes may represent a serious public health threat. Although MDR of S. pneumoniae isolates

with both erm(B) and mef(A) genes is documented, it is not known why they confer high MDR. Instead, it has been suggested that mutators are associated with the emergence of antimicrobial resistance in several pathogenic

bacterial species such as Escherichia coli, Pseudomonas aeruginosa, Neisseria meningitidis, Helicobacter pylori, and Staphylococcus aureus (Chopra et al., 2003). Mutators (hypermutable strains) are defined as bacterial strains with greater than normal mutation frequencies. Mutators are generally defective in the methyl-directed mismatch repair system, with mutations in mutS or mutL genes (Oliver et al., 2000). The relationship between antimicrobial resistance and frequency of mutation in S. pneumoniae has been investigated (Morosini et al., 2003; del Campo et al., 2005; Gould et al., 2007). However, whereas most studies have focused on fluoroquinolone resistance and point mutations Ribonucleotide reductase in hypermutable S. pneumoniae, the present study investigated the relationships between the presence of macrolide resistance determinants and the recombination rate. A total of 89 S. pneumoniae isolates were collected in a tertiary-care hospital in Korea, and antimicrobial susceptibility testing was performed. In addition, we determined erythromycin resistance determinants, erm(B) and mef(A) genes, by the duplex PCR method (Ko & Song, 2004). Of these, 46 S. pneumoniae isolates were selected and used for further research. Thirty-five isolates were erythromycin-resistant and the others were erythromycin-susceptible. Of the 27 erythromycin-resistant S.

No TNF-α, IL-1β or IL-10 was detected in the cochlear perilymph after the loss of most auditory hair cells, indicating the absence of severe inflammation. In contrast, Selleckchem Autophagy inhibitor we observed a significant and temporary increase in the level of extracellular high mobility group box 1 (HMGB1), a late mediator of inflammation that also functions as a signal of tissue damage. This increase coincided with epithelial remodelling of the injured organ of Corti, and occurred concomitantly with robust and transient cytoplasmic expression of acetylated HMGB1 within the non-sensory supporting cells,

Deiters cells. Here, HMGB1 was found to be enclosed within vesicles, a number of which carried the secretory vesicle-associated membrane-bound protein Rab 27A. In addition, transient upregulation of receptor for advanced glycation end-products (RAGE), an HMGB1 membrane receptor, was found in most epithelial cells of the scarring organ of Corti when extracellular levels of HMGB1 were at their highest. Altogether, these results strongly suggest that, in stressful conditions, Deiters cells liberate HMGB1 to regulate the epithelial reorganization of the injured organ of Corti through engagement of RAGE in neighbouring epithelial cells. “
“Previous results point towards

a lateralization of dorsolateral prefrontal cortex (DLPFC) function in risky decision making. While the right hemisphere seems involved in inhibitory cognitive control of affective impulses, the left DLPFC is crucial in the deliberative processing of information p38 kinase assay relevant for the decision. However, a lack of empirical evidence precludes definitive conclusions. The aim of our study was to determine whether anodal transcranial direct current stimulation (tDCS) over the right DLPFC with cathodal tDCS over the Selleck Metformin lDLPFC (anodal right/cathodal left) or vice versa (anodal left/cathodal right) differentially modulates risk-taking

in a task [the Columbia Card Task (CCT)] specifically engaging affect-charged (Hot CCT) vs. deliberative (Cold CCT) decision making. The facilitating effect of the anodal stimulation on neuronal activity was emphasized by the use of a small anode and a big cathode. To investigate the role of individual differences in risk-taking, participants were either smokers or non-smokers. Anodal left/cathodal right stimulation decreased risk-taking in the ‘cold’ cognition version of the task, in both groups, probably by modulating deliberative processing. In the ‘hot’ version, anodal right/cathodal left stimulation led to opposite effects in smokers and non-smokers, which might be explained by the engagement of the same inhibitory control mechanism: in smokers, improved controllability of risk-seeking impulsivity led to more conservative decisions, while inhibition of risk-aversion in non-smokers resulted in riskier choices.

After the membrane was blocked for 20 min in the blocking buffer (1% casein, 0.1 M maleic acid, 0.1 M NaCl, pH 7.5), the membrane was incubated with 0.1% streptoavidin-horseradish peroxidase conjugate (HRP; Sigma) in the blocking buffer for 20 min with gentle shaking. The membrane was washed four times with the washing buffer (0.3% Tween 20, 0.1 M maleic acid, 0.1 M NaCl, pH 7.5) for 5 min, followed by equilibration with the maleic acid buffer (0.1 M SD-208 research buy maleic acid, 0.1 M

NaCl) for 5 min with gentle shaking. The membrane was put on a clean sheet of plastic wrap and the light emitted by the DNA fragments produced on incubation in Chemi-Lumi One (Nacalai tesque, Kyoto, Japan) was recorded with LAS-4000 EPUVmini (Fuji Film, Tokyo, Japan). The molecular mass of the recombinant PyrR was determined by HPLC with

a size-exclusion chromatograph (Shodex Protein KW-803). A calibration curve was obtained based on the elution pattern of standard proteins as described previously (Yokochi et al., 2009). The subunit molecular mass was determined by SDS-PAGE as described previously (Yokochi et al., 2009). The primary sequence of the mll6786 gene product was homologous to several repressor proteins. The DMS12804 protein in Bordetella petrii showed the highest identity, 39%; the IP32953 protein in Yersinia pseudotuberculosis, 37%; and the Ymp protein in Pseudomonas mendocina, 37%. On the basis of this, mll6786 might encode a repressor protein and the gene product was designated as PyrR. The secondary structure of the PyrR protein was predicted with

the jpred 3 server (http://www.compbio.dundee.ac.uk/www-jpred/). The PyrR http://www.selleckchem.com/products/Trichostatin-A.html protein had an HTH motif: the Interleukin-2 receptor amino acid residues from V14 to S28 formed the first α-helix; those from E39 to L46, the second α-helix; and those from P51 to A62, the third α-helix. The α-helices were followed by two β-sheets (I66-V69 and G73-P77). The arrangement of the secondary structures in the PyrR protein was quite similar to that in a DNA-binding protein (YP_298823.1, PDB entry 3IHU) from Ralstonia eutropha JMP 134. A strain of M. loti in which the mll6786 gene was inactivated by insertion of a tetracycline resistance gene, was constructed and isolated as described in Materials and methods. PCR of the chromosome of the disruptant strain did not give a DNA band corresponding to the size of mll6786. Instead, it produced a DNA band corresponding to the size of the mll6786::Tc gene (Fig. 2a). Thus, an mll6786-disruptant strain was successfully prepared. The mll6786-disruptant strain grew as well as the wild-type strain in TY medium, but other phenotypic characteristics were not examined. If PyrR is a transcriptional repressor like the VanR subgroup proteins, the regulated enzyme activities in the mll6786-disruptant cells would be expected to increase following disruption of the pyrR gene. The enzyme activities in crude extracts of the wild-type and mll6786-disruptant M.

Animals were deeply anesthetized with ketamine and submitted to neurophysiological evaluation by electromyography of the mandibular branch of the facial nerve aiming at obtaining check details compound muscle action potentials (CMAPs). Outcome variables were the CMAP amplitude and latency values. To obtain the CMAPs, we used a portable electromyography system (Neuro-MEP-Micro®, Neurosoft, Dhaka, Bangladesh) connected to a battery-operated Pavilion dv5C portable personal computer (Hewlett-Packard). The Neuro-MEP.NET software (version 2.4.23.0, Neurosoft) was employed to assess the CMAP data obtained under the following configuration of the electromyography

system: 10-Hz high-pass filter, 10-kHz low-pass filter, notch filter off, 60 mV of leading edge signal, and 10-kHz of sampling rate. The electromyography protocol has been established specifically for

evaluation of the rat facial nerve and described in detail by Salomone et al. (2012). Histomorphometric analyses were performed blindly six weeks after surgical procedure, and this method was well established by Costa et al., 2006, Costa et al., 2007 and Costa et al., 2012. After sacrifice, the surgically repaired portion of the facial nerve was cut into four parts, two distally and two proximally related to the graft. One pair of proximal (middle find more of the autografting) and distal (3 mm distal to autografting) sections was fixed in 2% glutaraldehyde and 1% paraformaldehyde in 0.0031 M phosphate buffer, pH 7.3. After 60 min. in solution A, the tissue was postfixed for 2 h in 2% osmium tetroxide in phosphate buffer, dehydrated in ethanol, infiltrated

in propilene oxide and included in Epoxi® resin (Burlington, VT) until polymerization. Transversal, 1-μm sections were made and stained with 1% toluidine blue. Histological observations were carried out using light microscopy (Nikon Eclipse E 600, Nikon, Japan). The slides were photographed with a digital camera (Nikon Coolpix E 955, Nikon, Japan), and cell measurement taken (Sigma Scan Pro 5.0 software, SPSS Science). Qualitative analyses were performed according to general nerve architecture, pattern of tissue organization and myelination. For quantitative analyses of distal portion of the facial nerve, axons were counted in Cediranib (AZD2171) a partial area of 9.000 μm2 in three random microscopic fields for every fiber displaying its center within it. Total axon density was obtained by the ratio between total axon number and area. The shortest external diameter (including the myelin sheath) of all axons within a partial, randomly selected area (3.000 μm2) of the transversal section of the nerve was measured to evaluate the maturation of myelinated fibers (Mayhew and Sharma, 1984). The second pair of proximal and distal sections was fixed in 4% paraformaldehyde in phosphate-buffered saline.

1067G > A (p.G356D). This mutation has previously been reported in other FOP variant patients [7] and [25]. The R206H mutation may cause all three clinical types of FOP including classic FOP, FOP-plus and FOP variants. In this large patient series, all classic FOP and FOP-plus patients and one FOP variant carried the R206H mutation. Two FOP variant cases had non-R206H mutations. This phenomenon is consistent with a previous report [7] which only detected

non-R206H mutations in variant FOP patients. None of the 98 unaffected controls, including parents and siblings, had mutations in ACVR1. Penetrance of the ACVR1/ALK2 mutation was 100%. The parents of the FOP patients could recall the onset and features of flare-ups in all cases. In this study, the onset of FOP was considered to be the time when the first spontaneous flare-up appeared or the first HO lesion emerged after trauma. Sixty-nine percent of patients (50/72 cases) experienced the spontaneous Selleckchem E7080 onset of flare-ups. Thirty-six percent of patients (18/50 cases) experienced the spontaneous onset of a flare-up prior to two years of age; 58% of patients (29/50 cases) experienced the spontaneous onset of a flare-up between two and ten years of age; and 6% of patients (3/50 cases) experienced the spontaneous onset of a flare-up after age 10.

There Sotrastaurin in vivo was no significant difference between male and female patient’s distributions among various onset ages (Table 2). No patient with spontaneous onset of FOP had any premonitory signs or symptoms prior to the onset of a flare-up. The signs and symptoms accompanying the onset of a flare-up were different at different anatomic sites. If the flare-up was in the head, neck or trunk, the onset was usually acute with large painless or painful soft masses appearing within twelve hours. If the flare-up involved the extremities, patients were more likely to have had focal pain with decreased range of motion as their Unoprostone initial complaint, with or without the appearance of soft tissue swelling. Fifty-two percent of patients (26/50 cases) who experienced spontaneous onset of flare-ups presented with soft tissue swellings in the occipital region. Typically, as one mass subsided,

another one emerged and sequentially spread toward the back of the neck and trunk. Most masses eventually ossified, but some resolved completely. Twenty-three of the 26 patients who had spontaneous occipital masses had radiographic evidence of HO in the occipital and posterior neck regions at the first visit to our clinic, but three of the 26 patients who had reported flare-ups in the occipital region had no radiographic evidence of HO in the occipital region, although these three patients had HO at other sites where intercurrent flare-ups had occurred. Forty percent of patients (20/50 cases) with spontaneous onset of FOP presented with soft tissue swelling or focal edema in the neck, back, trunk or shoulder, and all of the soft tissue masses become ossified.

8 μL of this suspension was added to 200 mL of liquid medium and incubated in a shaker (200 rpm) bath for 16 h at 30 °C. The culture was then centrifuged at 3000 x g for 5 min at 4 °C, the supernatant was discarded and 20 mL of Milli-Q water was added to the pelleted cells, which were suspended and centrifuged again. This process was repeated three times. Finally the yeast cells were resuspended

in 3 mL of Milli-Q water. Aliquots of 107 cells were pre-incubated for 30 min in 800 μL of protein or peptide samples in 10 mM Tris–HCl, Bcl-2 inhibitor pH 6.0 or the dilution buffer alone. After pre-incubation, 200 μL of 500 mM glucose was added to the cells and the medium pH was measured every minute for 30 min. The amount of H+ released by the cell was calculated as the difference between the initial pH and the final pH (ΔpH), considering the equation pH = −log [H+]. The values are averages of triplicates for each experiment. The permeabilization of the plasma membrane was assessed by measuring absorption of SYTOX Green (Invitrogen, Grand Island, NY, USA) as described by [22]. This dye forms a fluorescent complex with nucleic acids, entering cells when the integrity of their plasma membrane is compromised. Fungal

cells were incubated with different concentrations of the test samples for 24 h and then exposed to 0.2 μM SYTOX Green for 30 min at room temperature. Obeticholic Acid order The cells were observed under a microscope (Axioskop 40 – Zeiss) equipped with a filter for fluorescein detection (excitation wavelength 450–490 nm and emission 500 nm). Fluorescent probes (LIVE/DEAD® Yeast Viability Kit – Invitrogen, Grand Island, NY, USA) were used to evaluate the viability and metabolic activity of yeasts in the presence of test samples. The yeast cells were grown overnight (16 h) in Sabouraud medium at 28 °C in the presence of either JBU, Jaburetox, dialysis buffer, or H2O2, and then centrifuged (3000 × g, 10 min) to remove the medium. Yeasts were suspended in buffer GH (2%

d-(+) glucose, 10 mM Na-HEPES, pH 7.2) and then 1 μM of FUN-1 and 12.5 μM of calcofluor were added. After more 2 h of incubation at 28 °C, the cells were viewed under a fluorescence microscope (Axioskop 40 – Zeiss) equipped with filters for different wavelengths to allow visualization of fluorescein (green), rhodamine (red) and DAPI (blue). Liothyronine Sodium Alternatively, cells were grown overnight in Sabouraud medium at 28 °C in the absence of test samples, followed by centrifugation to remove the medium. Yeasts were suspended in buffer GH (2% d-(+) glucose, 10 mM Na+-HEPES, pH 7.2). 100 μL of cell suspension were mixed with the samples JBU, Jaburetox, dialysis buffer, or H2O2, and maintained for 2 h at 28 °C. Then, 1 μM of FUN-1 and 12.5 μM of calcofluor were added and after 2 h at 28 °C, the cells were viewed under the microscope Axioskop 40 – Zeiss with different filters, as described above. All experiments were run in triplicates. Data were evaluated using “one-way” ANOVA followed by the t test of Bonferroni or Dunnett.

, Minneapolis, MN, USA) in DPBS containing 1% normal

donkey serum (Sigma, St. Louis, MO, USA) and 1% bovine serum albumin (BSA, Sigma). Next, coverslips were washed twice in DPBS and incubated with Alexa Fluor 546 goat anti-rat IgG (Invitrogen™, Life technologies) for 2 h at RT in the dark. After washing twice with DPBS, nuclear counterstain was performed by incubation with 0.5 μM de SYTO® 21 green fluorescent nucleic acid stain (Invitrogen™, Life Technologies) in DPBS for 2 min at RT. Coverslips were mounted in the ProLong® Gold antifade reagent (Molecular Probes®, Invitrogen™, Selleckchem Nutlin3a Life Technologies). The subcellular localization in dental pulp cells was visualized using the Leica TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany). Negative Target Selective Inhibitor Library in vitro controls were performed using the incubation buffer with no primary antibody. For extraction of total proteins, primary dental pulp cells, at passage five, from probands A and B (genotype: p.[N440del];[R152C]) and four control individuals (native TNAP) were seeded in 100 mm tissue culture dishes (40 × 104 cells per plate) in Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen™, Life Technologies) with 10% FBS for 24 h. Next, medium was changed to 5% FBS supplemented with 50 μg/mL ascorbic acid (AA) and 10 mM β-glycerol phosphate (βGP) for 7 days, with medium changed every other day. Cells were washed twice with DPBS, harvested

in ice-cold DPBS containing protease inhibitor cocktail (Sigma) and centrifuged at 850 g. The cell pellet was lysed with RIPA buffer (Sigma) containing protease inhibitor cocktail Demeclocycline (Sigma). Total protein concentration was determined by the Bradford method. Similar amounts of total protein from each sample (~ 70 μg) were resolved on 10% SDS-polyacrylamide gel electrophoresis

(PAGE) and then transferred to Hybond-ECL nitrocellulose membrane (GE Healthcare). Blots were blocked by incubation with 3% BSA in Tris buffer saline (TBS, pH 7.6) for 1 h. To detect the target protein, blots were probed with human alkaline phosphatase/ALPL rat monoclonal antibody (1:500, R&D Systems, Minneapolis, MN, USA) and secondary antibodies conjugated to horseradish peroxidase (1:30,000, ECL Anti-Rat IgG, GE Healthcare) in TBS containing 0.1% Tween 20. All steps of the incubation were performed for 1 h at room temperature with gentle agitation. The antigen–antibody complexes were detected by chemiluminescence using SuperSignal® West Fento Maximum Sensitivity Substrate (Thermo Scientific, Pierce Biotechnology, Rockford, IL, USA) for 1 min. Then, chemiluminescent images were acquired using an acquisition and documentation system (MicroChemi 4.2 from DNR Bio-Imaging Systems, Israel). Blots were re-probed with α-tubulin mouse monoclonal antibody (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and secondary ECL Anti-Mouse IgG antibodies, (1:20,000).