FIG. 3.

Effect of p66Shc regulation on glucose-induced Rac1 activation and its binding with Sos1. Cells transfected with p66-siRNA, and incubated in 20 mM glucose for 96 h, were for analyzed for (a) Rac1 binding with Sos1 by immunoprecipitating Rac1, followed by Western blotting for Sos1 (Supplementary Fig. S2). Supplementary Figure S2 for uncropped Sos1 and Rac1 Western blots from three different samples. (b) Cellular colocalization of Rac1 with Sos1 was determined by immunofluorescence using Texas Red (red) and Alexa-Flour 488 (green) conjugated secondary antibody, respectively. Fluorescence intensity for colocalized Rac1 and Sos1 was quantified using AxioVision Rel. 4.8 imaging analysis software. (c) Sos1 transcripts were quantified by qPCR using β-Actin as an internal control. The values obtained from cells in 5 mM glucose are considered as 1 (or 100%), and are represented as mean ± SD from —three to five samples per group. Five millimolars and 20 mM represent 5 or 20 mM glucose; l-Glu represents 20 mM l-glucose; p66-si or SC represents cells transfected with p66Shc siRNA or scrambled RNA, respectively, and incubated in 20 mM glucose for 96 h; 5+Sos-si represents cells transfected with Sos1 siRNA and incubated in 5 mM glucose. *p < 0.05 versus 5 mM glucose and ^#p < 0.05 versus 20 mM glucose. Sos1, Son of Sevenless 1.