Figure 7.

Tyrosine phosphorylation of Munc18c maps to a motif centered at Tyr219 in adipocytes. Fully differentiated 3T3-L1 adipocytes were preincubated in serum-free media for 2 h followed by treatment with freshly prepared 0.1 mM pervanadate (pV) for 5 min. (A) Cells were immediately fixed and permeabilized for immunostaining with a phosphospecific anti–p^Y219-Munc18c peptide antibody in the absence of a competitive peptide (image 1). The specificity of the antibody for the Y219-centered phosphopeptide was assessed by competition with the addition of 500 ng of the antigenic peptide in its nonphosphorylated (Non–P-Tyr, image 2) or phosphorylated (P-Tyr, image 3) state. Image 4 validates the presence of cells in the field imaged in image 3. Immunofluorescent confocal microscopy was used to visualize ≥50 cells each. Bars, 10 µm. (B) Cleared detergent lysates were prepared for anti-Munc18c immunoprecipitation (IP). Protein was resolved on 10% SDS-PAGE for immunoblotting (IB) with anti–p^Y219-Munc18c antibody. Ponceau S staining shows equal loading of the immunoprecipitation reactions. All data are representative of two to three independent experiments.