Figure 8.
ARHGAP17 interacts with CIP4 to regulate invadopodia. (A) Representative Western blot of CIP4 expression in SUM159 cells expressing non-targeting (CTRL) or CIP4-specific shRNAs. (B) Representative micrographs of CTRL and CIP4 KD (shRNA #4) SUM159 cells. F-actin and cortactin staining were used as markers for invadopodia. Cells were treated with PDBu for 30 min before fixation and staining. Arrowheads highlight invadopodia clusters. The zoomed regions are marked by a dashed line (scale bar, 20 μm; inset scale bar, 5 μm). (C) Percentage of cells with invadopodia in CTRL and CIP4 KD SUM159 cells treated PDBu for 30 min. Results are expressed as mean ± SEM of three independent experiments in which at least 200 cells per condition per experiment were counted. Significance was tested with a two-tailed t test: *, P < 0.05; **, P < 0.01. (D) Representative micrographs of SUM159 cells treated with PDBu for 30 min and stained for F-actin and endogenous CIP4. The zoomed region is marked by a dashed line (scale bar, 10 μm; inset scale bar, 2 μm). (E) Radial intensity analysis of F-actin and CIP4 in invadopodia. Lines are expressed as mean ± SEM of three independent experiments in which at least 10 invadopodia per condition per experiment were measured. (F) Western blot of co-immunoprecipitation of endogenous ARHGAP17 and CIP4 in SUM159 cells. (G) Western blot of GFP-pulldown of myc-CIP4 with GFP, ΔC ARHGAP17-GFP, or WT ARHGAP17-GFP. (H) Western blot of affinity precipitation of myc-CIP4 and CD ARHGAP17-GFP using GST-Cdc42-Q61L bound to Glutathione Sepharose 4B. In F–H, % input refers to the total lysate loaded in the gel relative to the amount used for the immunoprecipitation/pulldown. For all blots, molecular weights are indicated in kD. Source data are available for this figure: SourceData F8.

ARHGAP17 interacts with CIP4 to regulate invadopodia. (A) Representative Western blot of CIP4 expression in SUM159 cells expressing non-targeting (CTRL) or CIP4-specific shRNAs. (B) Representative micrographs of CTRL and CIP4 KD (shRNA #4) SUM159 cells. F-actin and cortactin staining were used as markers for invadopodia. Cells were treated with PDBu for 30 min before fixation and staining. Arrowheads highlight invadopodia clusters. The zoomed regions are marked by a dashed line (scale bar, 20 μm; inset scale bar, 5 μm). (C) Percentage of cells with invadopodia in CTRL and CIP4 KD SUM159 cells treated PDBu for 30 min. Results are expressed as mean ± SEM of three independent experiments in which at least 200 cells per condition per experiment were counted. Significance was tested with a two-tailed t test: *, P < 0.05; **, P < 0.01. (D) Representative micrographs of SUM159 cells treated with PDBu for 30 min and stained for F-actin and endogenous CIP4. The zoomed region is marked by a dashed line (scale bar, 10 μm; inset scale bar, 2 μm). (E) Radial intensity analysis of F-actin and CIP4 in invadopodia. Lines are expressed as mean ± SEM of three independent experiments in which at least 10 invadopodia per condition per experiment were measured. (F) Western blot of co-immunoprecipitation of endogenous ARHGAP17 and CIP4 in SUM159 cells. (G) Western blot of GFP-pulldown of myc-CIP4 with GFP, ΔC ARHGAP17-GFP, or WT ARHGAP17-GFP. (H) Western blot of affinity precipitation of myc-CIP4 and CD ARHGAP17-GFP using GST-Cdc42-Q61L bound to Glutathione Sepharose 4B. In F–H, % input refers to the total lysate loaded in the gel relative to the amount used for the immunoprecipitation/pulldown. For all blots, molecular weights are indicated in kD. Source data are available for this figure: SourceData F8.

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