In addition, when GPHN.FingR-GFP was expressed in either excitatory or inhibitory neurons it appeared in puncta adjacent to or overlapping presynaptic terminals labeled for GAD-65 (Figure S1 available online). These results are consistent with FingRs binding at high affinity to PSD-95 or Gephyrin. To corroborate the results from colocalization experiments we used biochemical

means to test for interaction between each FingR and its endogenous target protein in cortical neurons in culture. cDNAs encoding each FingR were incorporated into a lentivirus, which was used to infect the cultures. After expression of either PSD95.FingR-GFP or GPHN.FingR-GFP for 96 hr, we collected cell lysate, which was exposed to immobilized anti-GFP antibody. The immunoprecipitated protein complexes were blotted and stained for the presence of the endogenous target proteins. In cells infected with PSD95.FingR-GFP, the anti-GFP Obeticholic Acid purchase antibody coimmunoprecipitated a band at 95 kD that was labeled by the anti-PSD-95 antibody (Figure 2G), but the precipitate was not labeled with anti-Gephyrin antibodies. In cells where GPHN.FingR-GFP was expressed, the precipitate pulled down by the anti-GFP antibody contained a band at 80 kD that was labeled with the anti-Gephyrin antibody, but the precipitate was not labeled with the anti-PSD-95 antibody (Figure 2G). The GK domain of PSD95, which is contained within the target of the

PSD95.FingR selection, interacts with guanylate kinase-associated protein (GKAP), a protein that links PSD-95 to Shank-Homer complexes (Naisbitt et al., 1999; Tu et al., INK 128 1999) and has been implicated in synaptic remodeling (Shin et al., 2012). To determine whether binding of PSD95.FingR with PSD-95 interferes with

the interaction between PSD-95 and GKAP, we asked whether GKAP could pull down both PSD-95 and PSD95.FingR-GFP when all three proteins were expressed in COS cells. We found that, indeed, immunoprecipitation of GKAP resulted in coprecipitation of both PSD-95 and PSD95.FingR-GFP (Figure S1). Furthermore, in COS cells expressing only GKAP and PSD95.FingR-GFP, immunoprecipitation of GKAP did not cause coprecipitation of PSD95.FingR-GFP. Resminostat Thus, in the GKAP/PSD95/PSD95.FingR-GFP complex, GKAP and PSD95.FingR must both bind to PSD95, indicating that binding of PSD95.FingR to PSD-95 does not disrupt binding of PSD-95 to GKAP. In order for FingRs to accurately report the localization and trafficking of their endogenous targets, it is necessary to minimize the excess, unbound FingR. To demonstrate the effect of overexpressing FingRs, we expressed either GPHN.FingR-GFP (Figures 3A–3C) or PSD95.FingR-GFP (Figure S2) for periods of 48 hr to 6 days and found that each appears in a diffuse pattern consistent with the signal from the excess, unbound FingR overwhelming the signal from the FingR bound to its target (Figures 3A–3C, Figure S2).