Figure 3.

pV induces IR–Munc18c complex formation and Munc18c–syntaxin 4 dissociation. (A–C) Cleared detergent lysates prepared from fully differentiated 3T3-L1 adipocytes that were incubated in serum-free medium for 2 h and then treated with or without 0.1 mM of freshly prepared pervanadate (pV) for 5 min were used in coimmunoprecipitation (IP) reactions: anti-Munc18c (A), anti-IR (B), or anti–syntaxin 4 (C, Syn4). Anti-Munc18c and anti–syntaxin 4 immunoprecipitation reactions were processed in parallel from the same starting lysates, which were confirmed to contain equivalent Munc18c protein (lysates). Munc18c and syntaxin 4 abundances in corresponding starting lysates used for B were also confirmed (Lysates). Coimmunoprecipitated proteins were resolved on 10% SDS-PAGE for immunoblotting (IB) with anti-Munc18c, anti-IR, and anti–syntaxin 4 antibodies; the Munc18c blot was stripped and reprobed with antiphosphotyrosine 4G10 (P-Tyr). Data represent the means ± SEM from three independent experiments for each type of immunoprecipitation, quantified as the ratio of IR/total Munc18c (A, i), tyrosine-phosphorylated Munc18c/total Munc18c (A, ii), or Munc18c/total syntaxin 4 (C) immunoprecipitated, with each set normalized to untreated = 1.0; *, P < 0.05 versus untreated. Black lines indicate that intervening lanes have been spliced out.