Figure 4

Tyrosine phosphorylation of Munc18c regulates its binding to syntaxin4 and PTP1B. A) Munc18c was immunoprecipitated from lysates of wild type adipocytes and adipocytes with knockdown and reconstituted expression of Munc18c (R and Y/F mutants) at basal and insulin-stimulated conditions, then immunoblotted using antibodies for phosphotyrosine, PTP1B, Syn4 and Munc18c. Bar graphs represent normalized data for tyrosine phosphorylated Munc18c/Munc18c and Syn4/Munc18c and presented as means ± SEM. B) Syn4 was immunoprecipitated from lysates of WT adipocytes and adipocytes with knockdown and reconstituted expression of Munc18c (R and Y/F mutants) at basal and insulin-stimulated conditions, then immunoblotted using antibodies for Munc18c, Glut4 and Syn4. Bar graphs represent normalized data for Munc18c/Syn4 and Glut4/Syn4 and presented as means ± SEM. (*; P ≤ 0.05, **; P ≤ 0.01) indicate significant difference between insulin stimulated and non-stimulated cells, and (#; P ≤ 0.05, ##; P ≤ 0.01) indicate significant difference between different cells versus WT for the corresponding treatment.