Figure 3

Munc18c is a PTP1B substrate. A) PTP1B was immunoprecipitated from lysates of PTP1B KO adipocytes and KO adipocytes reconstituted with PTP1B D/A at basal and insulin-stimulated conditions then immunoblotted using antibodies for Munc18c and PTP1B. B) Munc18c knockdown (KD) cells were reconstituted with Munc18c wild type (R) and Y/F mutants (Y^218/219F and Y⁵²¹F). Lysates were immunoblotted for Munc18c and Erk1/2. C) Sequences were aligned relative to amino acids Tyr^218/219 and Tyr⁵²¹ of mouse Munc18c. The boxed region denotes the location of amino acids Tyr^218/219 and Tyr⁵²¹ across species. D) 3T3-L1 wild type (WT) and Munc18c knockdown cells (reconstituted with Y/F mutants) were transiently transfected with PTP1B WT and D/A. PTP1B was immunoprecipitated using FG6 antibodies from starved and insulin-stimulated cells lysed in NP40 or RIPA (R), resolved using SDS-PAGE then immunoblotted using antibodies for Munc18c and PTP1B. Bar graph represents Munc18c levels in PTP1B immunoprecipitates normalized to level of PTP1B and presented as means ± SEM. (*; P ≤ 0.05, **; P ≤ 0.01) indicate significant difference between insulin stimulated and non-stimulated cells, (#; P ≤ 0.05, ##; P ≤ 0.01) indicate significant difference between Munc18c Y/F mutants and Munc18c WT insulin stimulated D/A cells, (&&; P ≤ 0.01) indicates significant difference between RIPA-treated and non-treated insulin stimulated WT cells, and (^^; P ≤ 0.01) indicates significant difference between vanadate-treated and non-treated insulin stimulated D/A cells.