JCS016881 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Interaction of endogenous IQGAP1, EXO70 and SEPT2 and effects of exogenous IQGAP1 domains. (Ai) Antibodies for endogenous IQGAP1 can precipitate endogenous EXO70 and SEPT2 from different breast cancer cell lines. Equal amounts of total cell lysates from the indicated cell lines were used for co-IP with IQGAP1 antibodies. EXO70 and SEPT2 antibodies were used for immunoblotting. LC indicates the light chain (∼25 kD) and mock is intersectin antibody used for control. Input indicates 5% of the lysate used for the IP probed with IQGAP1, EXO70 and SEPT2 antibodies. (Aii) IQGAP1, EXO70 and SEPT2 are cytosolic proteins. Lysate from β-cells was fractionated as described in the Materials and methods. Equal protein amounts from the cytosol and the membrane fractions were probed for IQGAP1, EXO70 and SEPT2. Actin was blotted as cytosol (C) and syntaxin as membrane (M) markers. (B) Exogenous V5-IQGAP1-N and V5-IQGAP1-IR-WW domains co-IP with endogenous EXO70 in β-Cdc42WT stable cell lines. β-cells stably expressing Cdc42WT were transiently transfected with the indicated IQGAP1 domains, co-IP with EXO70 antibodies was carried out and blotted with V5 antibodies. (C) Co-IP of endogenous EXO70 and SEPT2 with IQGAP1 antibodies in the presence of exogenous IQGAP1 domains. (i) Expression of the IQGAP1 constructs depicted in Fig. 3A, in COS7 cells, 5% of total lysate was blotted with V5 antibodies. (ii) Equal amounts of proteins from COS7 cells expressing the indicated IQGAP1 constructs in S1i were used to co-IP EXO70 and SEPT2 with 1 µg of IQGAP1 antibody. The blot is representative of blots obtained using antibodies for EXO70 for the IP or antibodies for the C- or N-terminus IQGAP1 for the IP. HC and LC indicate the heavy (50 kD) and the light (25 kD) chains of the antibody, respectively. (iii) Quantitation of endogenous IQGAP1 association with EXO70 in the presence of exogenous IQGAP1 and domains stably expressed in β-cells from blots similar to the one shown in S1ii for n=4 ± the s.d. from the mean.

• Supplemental Figure S2 -

  Fig. S2. Localization of IQGAP1 at the ER and the plasma membrane in β-cells. (A) Co-localization of IQGAP1 with the polarity ER-resident marker IP₃R-3 in β-cells. The upper panel shows co-localization of IQGAP1 (red) and the ER resident marker IP₃R-3 (green) in single cells at a paranuclear position indicating the ER. Cells were photographed at higher magnification with a 100×, NA 1.4 objective. The lower panel shows co-localization at the cell-cell contacts of islet-like cells photographed with a 60× objective. (B) Co-localization of IQGAP1 with the t-SNARE syntaxin 1A in β-cells. Fixed cells were double-stained with IQGAP1 (red) and syntaxin 1A (green) antibodies.

• Supplemental Figure S3 -

  Fig. S3. Endogenous IQGAP1 influences insulin secretion in pancreatic β-cells. (Ai) Depletion of IQGAP1 does not affect the expression levels of EXO70 or SEPT2 in β-cells. RNA 21-oligomers for IQGAP1-RNAi, scramble or non-targeting control, described in the Materials and methods section, were transiently transfected into β-cells at ∼50% confluency and immunoblotting performed after 48 hours with the indicated antibodies. (Aii) Expression of wild-type and the RNAi-refractory IR-WW (IR-WW^R) constructs in β-cells. (B) Depletion of IQGAP1 decreases the induced level of secreted insulin in β-cells. Cells transiently transfected with scramble control (first column), IQGAP1-RNAi (middle column) or co-transfected with IQGAP1-RNAi+IR-WW^R plasmid (last column) encoding the RNAi-resistant construct of the IQGAP1 IR-WW domain were used for measuring glucose-induced insulin secretion.