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Supplemental material Jewell et al., http://www.jcb.org/cgi/content/full/jcb.201007176/dc1 T H E J O U R N A L O F C E L L B I O L O G Y Figure S1. IR Munc18c association is independent of IRS-1. (A and B) IRS-1 (A) or Munc18c (B) was immunoprecipitated (IP) from 3T3-L1 adipocytes stimulated with or without 100 nm insulin for 5 min. Proteins were subjected to 10% SDS-PAGE and transferred to PVDF for immunoblotting (IB) with anti IRS-1, anti-munc18c, and anti-ir antibodies. The presence of IRS-1 and Munc18c in the starting lysates used in A was confirmed by immunoblotting (Lysates). Data are representative of at least three independent sets of cell lysates. S1

Figure S2. The IR phosphorylates the IRS-1 peptide in vitro. The IRS-1 (Y608) peptide (NH2-KKHTDDGYMPMSPGVA-COOH substrate; Enzo Life Sciences) was incubated in the presence of -[ 32 P]ATP with or without the recombinant subunit of the IR in the same reaction buffer and spotted on filter paper. The peptide was then precipitated with 1% phosphoric acid, washed with acetone, and dried, and the incorporation of radioactivity was determined by scintillation counting. Data are representative of the means of two independent experiments, each performed in duplicate. The times on the bottom of the graph are given in minutes. S2

Figure S3. Comparable phenotypic changes in insulin-stimulated GLUT4-GFP translocation to the plasma membrane in nonpermeabilized and permeabilized 3T3-L1 adipocytes and the effects of RNAi-mediated depletion. (A C) In brief, 3T3-L1 adipocytes were electroporated, and after a 48-h incubation, cells were subjected to (A) detergent lysis for immunoblotting (IB), (B) incubation in serum-free medium for 2 h followed by stimulation with or without insulin for the measurement of glucose uptake using the protocol of Shi and Kandror (2008; data represent the means of two independent experiments, each performed in duplicate), or (C) stimulation with 100 nm insulin followed by fixation with or without permeabilization for immunofluorescent confocal microscopy. Cells expressing GLUT4-GFP were phenotyped as having a rim or no rim, with percentages showing the relative number as exhibiting rim per condition in the bottom left of each image. Data represent 155 181 cells imaged for each condition. Bars, 5 µm. Black lines indicate that intervening lanes have been spliced out. S3

Figure S4. Munc18c mutant expression and phosphomimetic Munc18c-binding characteristics. (A) Cells were coelectroporated with simunc18c and sirna-resistant pcdna3-flag-munc18c-wt or phosphodefective mutant DNAs as described in Materials and methods (ENDOG., endogenous Munc18c, nontransfected). Detergent cell lysates were prepared, and proteins were resolved on 10% SDS-PAGE for evaluation of knockdown efficiency and exogenous protein expression by anti-munc18c immunoblotting (IB). Clathrin was used for normalization. (B) Electroporated adipocytes expressing Flag-Munc18c- WT, -Y521E, -Y219E, or -Y219E/Y521E mutants were incubated in serum-free medium for 2 h stimulated with insulin for 5 min for subsequent detergent lysis and utilization in anti-flag immunoprecipitation (IP) reactions to detect association with endogenous IR by immunoblotting. Equivalent IR abundance in corresponding starting lysates was confirmed on a separate gel, with tubulin as a loading control (Lysates). Data are representative of at least three independent experiments using multiple DNA batches. (C) Binding of the phosphomimetic mutants to syntaxin 4 (Syn4) in CHO/IR cells as described for Fig. 8 C and in Materials and methods. Reactions with Y521E or the other two mutants (Y219E and Y219E/Y521E), each with a paired WT-positive control, were resolved on two separate gels. Syntaxin 4 expression in the corresponding starting lysates was confirmed on a separate gel, with tubulin as a loading control (Lysates). Data are representative of three to five independent experiments each. Black lines indicate that intervening lanes have been spliced out. S4

Reference Shi, J., and K.V. Kandror. 2008. Study of glucose uptake in adipose cells. Methods Mol. Biol. 456:307 315. doi:10.1007/978-1-59745-245-8_23 S5

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