Figure 5.

Tyrosine phosphorylation of Munc18c regulates deoxy-glucose uptake. A) Immunoblots of GLUT4 in cytosol and membrane fractions of differentiated WT adipocytes and adipocytes with knockdown (KD) and reconstituted expression of Munc18c (R and Y/F mutants) at basal and insulin-stimulated conditions. Gels were also immunoblotted for GST and IRE1α to determine purity of the cytosol and membrane fractions, respectively. B) Data are presented as mean ± SEM from four independent experiments. (*; P ≤ 0.05, **; P ≤ 0.01) indicate significant difference between insulin stimulated and non-stimulated cells in cytosol and membrane fractions, (#; P ≤ 0.05, ##; P ≤ 0.01) indicate significant difference between different cells versus WT for the corresponding treatement in the cytosol fraction, and (^; P ≤ 0.05, ^^; P ≤ 0.01) indicate significant difference between different cells versus WT for the corresponding treatment in the membrane fraction. C) Basal and insulin-stimulated 2-deoxy-[³H] glucose uptake was determined in differentiated WT adipocytes and adipocytes with KD and reconstituted expression of Munc18c at basal and insulin-stimulated conditions. Data are presented as mean ± SEM from five independent experiments. (*; P ≤ 0.05, **; P ≤ 0.01) indicate significant difference between the different cells versus WT at the corresponding treatment. Immunoblots of pAkt (T308 and S473) and Akt in lysates of differentiated WT adipocytes and adipocytes with KD and reconstituted expression of Munc18c at basal and insulin-stimulated conditions.