26 nM) of the unlabelled toxin for 1 hour at 37°C. Heterologous competitive binding assays Similar trends were observed for both crude Btj toxin and crude Bt 22 toxin (Figure 3). Both AMPK inhibitor graphs showed a decreasing trend in the percentage of biotinylated purified Bt 18 toxin bound to CEM-SS with increasing toxin concentrations. For crude Btj toxin the percentage of bound biotinylated purified Bt 18 toxin

significantly decreased from 100% to 78% at 59.26 nM (p < 0.001). For crude Bt 22 toxin, the percentage of bound biotinylated purified 3-MA Bt 18 toxin significantly decreased from 100% to 80.81% at 59.26 nM (p < 0.05). However, the difference between crude Btj toxin and crude Bt 22 toxin was statistically insignificant (p > 0.05). Figure 3 Heterologous competitive binding assays- biotinylated purified Bt 18 toxin versus crude Btj and crude Bt 22 toxins. Fixed concentration (7.41 nM) of biotinylated purified Bt 18 toxin was allowed to compete with various concentrations (0 nM to 59.26 nM) of crude Btj toxin and crude Bt 22 toxin separately for 1 hour at 37°C using CEM-SS cell line. It was observed that the graphs show a similar pattern for all the anticancer drugs i.e., the higher

the drug concentration, the lower the percentage of biotinylated purified Bt 18 toxin bound to CEM-SS cells (Figure 4). There was a statistically significant but minor decrease in the percentage of binding of the biotinylated toxin on CEM-SS cells when competed with methotrexate Avapritinib solubility dmso (< 30%, p < 0.05) and Ketotifen doxorubicin (< 10%, p < 0.05) with increasing drug concentration. On the other hand, it was found that cisplatin, etoposide and navelbine caused a greater decrease (> 30%) in the percentage of binding of the biotinylated toxin on CEM-SS with increasing drug concentration. This decrease was significant for cisplatin and etoposide (p < 0.001) but insignificant for navelbine (p > 0.05) at the highest drug concentration (59.26 nM). The percentage of displacement of the biotinylated toxin on CEM-SS at the highest drug concentration

for the cisplatin, doxorubicin, etoposide, navelbine and methotrexate were 32.76%, 9.82%, 44.67%, 40.27% and 20.40% respectively. However, such high percentage of displacement of the biotinylated toxin at the highest drug concentration was also confounded by a high percentage of cell death (results not shown). Therefore, displacement of the biotinylated toxin at the highest drug concentration may or may not be due to true competition. Figure 4 Heterologous competitive binding assays- biotinylated purified Bt 18 toxin versus various anticancer drugs. Fixed concentration (7.41 nM) of biotinylated purified Bt 18 toxin was allowed to compete with various concentrations (0 nM to 59.26 nM) of cisplatin, doxorubicin, etoposide, methotrexate and navelbine, separately for 1 hour at 37°C.