Figure 4

Immunoblot analysis after Co-immunoprecipitation assay. Immunoprecipitation assays of 14-3-3ζ, NFκB, β-catenin and Bcl-2 proteins were carried out in oral cancer cells, HSC2, as described in Materials and Method section. The immunoprecipitates were resolved on 12% SDS-PAGE and electro-transferred on PVDF membrane followed by blocking with 5% non-fat milk O/N. Blot was incubated with 14-3-3ζ antibody and protein expression was determined using Enhanced Chemiluminescence method. Figure 4a shows immunoblot analysis 14-3-3ζ, demonstrating the binding of 14-3-3ζ protein with NFκB, β-catenin and Bcl-2 proteins. However, no band of 14-3-3ζ was observed in the negative control. Similarly, reverse immunoprecipitation assays were carried out using specific antibodies for NFκB, β-catenin and Bcl-2 proteins. Figure 4b shows immunoblot analysis of: (i) NFκB; (ii) β-catenin and (iii) Bcl-2 protein confirming the binding of these proteins with 14-3-3ζ.