Fig 1: Actin and microtubule associated protein IQGAP1 localizes to actin coats on fused secretory vesicles. (A) IQGAP1 expression in ATII cells was measured with RT-PCR relative to the expression of the housekeeping gene HMBS immediately after isolation (day 0) and after 2 days of cell culture (day 2). (B) Western blot with a-IQGAP1 antibody with freshly isolated ATII cells (d0), after 1 day of culture (d1) and after 2 days of culture (d2). M = Molecular weight marker. Ponceau S staining was used as control for equal loading. Full-length gel is shown on Supplementary Fig. 7. (C) ATII cells were stimulated for secretion with ATP, fixed and immunolabelled for IQGAP1 and ABCa3. Actin was labelled with alexa fluor 568 phalloidin and cell nuclei with Hoechst 33342. Fused secretory vesicles (arrow and inserts) were identified by the presence of the actin coat. Overlay shows colocalization between IQGAP1, actin coat and secretory vesicle. Arrowheads: non-fused vesicles; scale bar: 10 µm, insert: 2 µm.
Fig 2: IQGAP1 translocates to secretory vesicles after fusion whereas IQGAP1 silencing decreased actin-GFP fluorescence on actin coats. (A) ATII cells were transfected with IQGAP1-GFP, actin-DsRed and stained with LTB. Arrow: fusing vesicle enlarged on B; scale bar: 10 µm. (B) Changes in IQGAP1-GFP, actin-DsRed and LTB fluorescence during exocytosis. Numbers indicate time; scale bar: 2 µm. (C) IQGAP1-GFP and actin-DsRed fluorescence intensity change on fusing vesicles (29 vesicles, 12 experiments and 5 cell isolations). Dashed lines on C, D, E: time of fusion. (D) IQGAP1-GFP fluorescence on fusing vesicles in cells treated with latrunculin, colchicine or nocodazole (31 vesicles, 8 experiments and 4 cell isolations (latrunculin); 28 vesicles, 7 experiments and 3 cell isolations (colchicine); and 21 vesicles, 11 experiments and 5 cell isolations (nocodazole)). The dotted line on D and E shows control IQGAP1-GFP fluorescence. (E) IQGAP1-GFP fluorescence on fusing vesicles in cells treated with B toxin or Rho-GTPase inhibitor (38 vesicles, 18 experiments and 3 cell isolations (B toxin); 24 vesicles, 8 experiments and 3 cell isolations (Rho inhibitor)). (F) ATII cells were transfected with IQGAP1-silencing siRNA or control siRNA and relative IQGAP1 expression was measured with RT-PCR (mean +/- SD, 3 cell isolations). (G) Immunoblotting with a-IQGAP1 three days after cell isolation and transfection with IQGAP1-silencing siRNA (si) or control siRNA (c). Ut: untreated control. Full-length gel is on Supplementary Fig. 8. (H) Cells transfected with IQGAP1-silencing siRNA or control siRNA were co-transfected with actin-GFP, stimulated for exocytosis and imaged for actin coat formation. N: nucleus; arrows: actin coats; scale bar: 10 µm. (I) Actin-GFP fluorescence on actin coats was measured as described on Fig. 4. Numbers indicate the number of measured actin coats (9 experiments and 3 cell isolations (control siRNA), 7 experiments and 3 cell isolations (IQGAP1 siRNA)). ***P < 0.001; two-tailed t-test. (J) Compression of actin coats in cells transfected with IQGAP1 siRNA was measured as a decrease in actin coat diameter (16 vesicles, 8 independent experiments and 3 cell isolations (control siRNA); 19 vesicles, 5 independent experiments and 3 cell isolations (IQGAP1 siRNA)).
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