Supplemental Information-Baumann et.al. MS# S11770A

CAP Defines a Second Signaling Pathway Required for Insulin-Stimulated Glucose Transport – Supplemental Figure List (MS# S11770A)

Christian A. Baumann*, Vered Ribon*, Makoto Kanzaki‡, Debbie C. Thurmond‡, Silvia Mora‡, Satoshi Shigematsu‡, Perry E. Bickel†, Jeffrey E. Pessin‡ and Alan R. Saltiel*

*Department of Physiology, University of Michigan School of Medicine, Ann Arbor, Michigan 48109, USA and Department of Cell Biology, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, MI 48105, USA

‡Department of Physiology & Biophysics, The University of Iowa, Iowa City, IA 52242, USA

†Department of Internal Medicine and Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA

Supplemental Figure 1.Plasma membrane localization of CAP, CAPSH3 and caveolin I in 3T3-L1 adipocytes. 3T3-L1 adipocytes were electroporated with 50g DNA containing CAP or CAPSH3 as described in Methods. Transfected adipocytes were paraformaldehyde fixed and stained with anti FLAG (Sigma) and caveolin I (Transduction Laboratories) antibodies. Cells were then incubated with FITC and Texas Red conjugated anti-mouse and anti-rabbit antibodies respectively (Vector Laboratories). Representative confocal immunofluorescence images of anti-FLAG-FITC stained CAP (panels 1) or CAPSH3 (panel 2)expressing adipocytes with anti-caveolin Texas Red labeling for endogenous caveolin I (panels 3 and 4). The merge of the FITC labeled CAP and CAPSH3 with Texas Red labeled caveolin I are shown in panels (5) and (6), respectively.

Supplemental Figure 2.Caveolin and flotillin co-localize in plasma membrane subdomains (rosettes) in 3T3-L1 adipocytes. Plasma membrane sheets were prepared as described under Methods. Representative confocal immunofluorescence images of plasma membrane sheet localized FITC-flotillin (panels 1 and 2) and Texas Red- caveolin (panels 3 and 4) isolated from basal (panels 1 and 3) or insulin-treated cells (panels 2 and 4). The co-localization of FITC-labeled flotillin and Texas Red-labeled caveolin are shown in panels (panels 5 and 6), respectively.

Supplemental Figure 3. Insulin stimulates the translocation of Cbl to caveolae-enriched membranes in 3T3-L1 adipocytes. Control and insulin-stimulated 3T3-L1 adipocytes were non-detergent homogenized, sonicated and subjected to sucrose gradient fractionation. These panels are representative immunoblots for caveolin, flotillin and Cbl in the isolated fractions from low to high density (fraction 1-12).

Supplemental Figure 4. CAP and CAPSH3 have no effect on GLUT1 translocation in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocyte nuclei were co-microinjected with 0.2mg/ml of MBP-Ras plus either the CAP or CAPSH3 cDNA. The cells allowed to recover for 24hours, and then treated without (panels 1 and 2)or with 100nM insulin (panels 3-6) for 30min. Plasma membrane sheets were prepared and MBP-Ras was detected with a FITC labeled anti-MBP antibody (panels 1, 3 and 5) and GLUT4 with a Cy5 labeled anti-GLUT4 antibody (panels 2, 4 and 5). These are representative images obtained from five independent experiments and visualization of 60-91 individual plasma membrane sheets.