Figure 6. The GAP activity of SH3BP1 is required for junction formation. (A) A431 cells were transfected with siRNAs, serum starved, and then stimulated with EGF for 5 min. After fixation, the cells were stained as indicated. Shown are epifluorescence images from the dorsal aspect of the cells. Note that A431 dorsal ruffles are positive for SH3BP1, β-catenin, and ZO-1, which are disrupted upon SH3BP1 depletion. (B) GFP-actin–expressing A431 cells were transfected with siRNAs, serum starved, and then stimulated with EGF. Shown are images of time-lapse recordings taken from the dorsal aspect of the cells. See also Videos 1, 2, 3, and 4. (C–F) A431 cells were transfected with the indicated siRNAs and, after 3 d, with RNAi-resistant cDNAs encoding HA-tagged wild-type or GAP-deficient SH3BP1. The cells were serum depleted, stimulated with EGF, and then analyzed by immunoblotting (C) and immunofluorescence (D–F). E shows a quantification of cells with junctional ZO-1 staining. Shown are means ± 1 SD, representing the cells in at least seven different fields per condition (n ≥ 7). Note that mutant SH3BP1 has a strong dominant-negative effect on the actin organization. Bars: (A, D, and F) 10 µm; (B) 5 µm.
Figure 6.

The GAP activity of SH3BP1 is required for junction formation. (A) A431 cells were transfected with siRNAs, serum starved, and then stimulated with EGF for 5 min. After fixation, the cells were stained as indicated. Shown are epifluorescence images from the dorsal aspect of the cells. Note that A431 dorsal ruffles are positive for SH3BP1, β-catenin, and ZO-1, which are disrupted upon SH3BP1 depletion. (B) GFP-actin–expressing A431 cells were transfected with siRNAs, serum starved, and then stimulated with EGF. Shown are images of time-lapse recordings taken from the dorsal aspect of the cells. See also Videos 1, 2, 3, and 4. (C–F) A431 cells were transfected with the indicated siRNAs and, after 3 d, with RNAi-resistant cDNAs encoding HA-tagged wild-type or GAP-deficient SH3BP1. The cells were serum depleted, stimulated with EGF, and then analyzed by immunoblotting (C) and immunofluorescence (D–F). E shows a quantification of cells with junctional ZO-1 staining. Shown are means ± 1 SD, representing the cells in at least seven different fields per condition (n ≥ 7). Note that mutant SH3BP1 has a strong dominant-negative effect on the actin organization. Bars: (A, D, and F) 10 µm; (B) 5 µm.

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