Figure 5.

Effect of Rap1b transfection on the high glucose–perturbed Bcl-2–Bax and Bcl-2–Rap1b interactions. (A) The protein–protein interactions were studied using IP followed by Western blotting. The antibodies were switched for each of the procedures, and perturbed interactions under high-glucose ambience (25 to 35 mM) were reflected by a decrease in the band density. These interactions seem to be normalized with restoration of the band intensity after Rap1b overexpression. (B) Band density averaged from duplicate experiments is included. (C and D) Rap1b interactions with mutant Bcl-2 GST proteins, reflecting deletion of BH1 through BH4 domains, were investigated. The deletion of BH4 domain of Bcl-2 leads to a loss of binding with Rap1b protein (lane 3), whereas it was unaffected with other mutant proteins, suggesting specificity of Bcl-2–Rap1b interactions.