Fig. 7.

Overexpression of Doc2b coordinately decreases Munc18c–Syn4 binding while increasing Syn4 activation in L6 GLUT4-myc myoblasts. Detergent lysates prepared from L6 GLUT4-myc myoblasts transfected to express GFP-tagged Doc2b or GFP alone and stimulated with insulin for 5 min were used in anti-Syn4 immunoprecipitation reactions and co-precipitated Munc18c or Doc2b proteins were detected by immunoblotting (a), or in GST-VAMP2 interaction assays for detection of the Syn4 present in lysates that is accessible to the exogenous GST–VAMP2 probe (b). Proteins were immunoblotted for Syn4, GST and GFP or GFP–Doc2b (∼75 kDa). Quantification is represented in the adjacent bar graphs. Data are representative of the average ± SE of three independent experiments of the ratio of Munc18c/Syn4 (%), and Syn4/GST–VAMP2 (fold), respectively; *p<0.05 vs GFP