Figure 8.
Tetraspanin CD81 is part of the JAM-A - CD9 - αvβ5 integrin complex and regulates Rac1 activity. (A) CoIP of αvβ5 integrin with JAM-A from CD9 KD MCF7 cells. Right: Expression of CD9 in control cells (MCF7-pLVTHM) and CD9 knockdown cells (MCF7-pLVTHM-CD9-shRNA). (B) CoIP of CD81 with JAM-A. (C) CoIP of CD81 with JAM-A mutant constructs: ΔD1-JAM-A (lacks D1 Ig domain), JAM-A/Δ3 (lacks PBM). Abbreviations: Fl, Flag. (D) CoIP of αvβ5 integrin with JAM-A from CD81 KD MCF7 cells and CD9/CD81 double KD MCF7 cells. Control KD and CD81 KD were achieved with OnTarget plus non-targeting pool and hCD81 OnTarget plus siRNA pool, respectively. (E) CoIP of αvβ5 integrin (left) and Src (right) with CD81. (F) Quantification of CIL after combined depletion of CD9 and CD81 in MCF7 cells. Number of collisions analyzed: n = 83 for Ctrl KD–WT collisions, n = 67 for CD9/CD81 KD–WT collisions (three independent experiments). (G) Top: GST-PD assay from CD81 KD MCF7 cells (pInducer10-mir-RUP-PheS-hCD81 shRNA) or from control MCF7 cells (pLVTHM). Bottom: Quantification of Rac1 activities in CD81 KD cells compared with control KD cells. Levels of active Rac1 (GTP-Rac1) are expressed as ratios of signal intensities of GTP-Rac1 to total Rac1. Data is derived from three independent experiments. (H) CD81 suppresses Rac1 activity at cell–cell contacts during cell collisions. Quantification of FRET ratios in Ctrl MCF7 (Ctrl KD) and CD81 KD MCF7 cells (CD81 KD) collided with wildtype MCF7 (WT) cells. Rac1 activity at cell–cell contacts relative to cell-free edges is depicted as the ratio of FRET signals (mean grey values) at cell–cell contacts to the FRET signals (mean grey values) at the cell-free edge of the Raichu-Rac1-expressing cell (n = 71 for each condition, three independent experiments. All statistical analyses shown in the figure were performed using unpaired Student’s t test. Data are presented as arithmetic means ± SD; NS, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Source data are available for this figure: SourceData F8.

Tetraspanin CD81 is part of the JAM-A - CD9 - αvβ5 integrin complex and regulates Rac1 activity. (A) CoIP of αvβ5 integrin with JAM-A from CD9 KD MCF7 cells. Right: Expression of CD9 in control cells (MCF7-pLVTHM) and CD9 knockdown cells (MCF7-pLVTHM-CD9-shRNA). (B) CoIP of CD81 with JAM-A. (C) CoIP of CD81 with JAM-A mutant constructs: ΔD1-JAM-A (lacks D1 Ig domain), JAM-A/Δ3 (lacks PBM). Abbreviations: Fl, Flag. (D) CoIP of αvβ5 integrin with JAM-A from CD81 KD MCF7 cells and CD9/CD81 double KD MCF7 cells. Control KD and CD81 KD were achieved with OnTarget plus non-targeting pool and hCD81 OnTarget plus siRNA pool, respectively. (E) CoIP of αvβ5 integrin (left) and Src (right) with CD81. (F) Quantification of CIL after combined depletion of CD9 and CD81 in MCF7 cells. Number of collisions analyzed: n = 83 for Ctrl KD–WT collisions, n = 67 for CD9/CD81 KD–WT collisions (three independent experiments). (G) Top: GST-PD assay from CD81 KD MCF7 cells (pInducer10-mir-RUP-PheS-hCD81 shRNA) or from control MCF7 cells (pLVTHM). Bottom: Quantification of Rac1 activities in CD81 KD cells compared with control KD cells. Levels of active Rac1 (GTP-Rac1) are expressed as ratios of signal intensities of GTP-Rac1 to total Rac1. Data is derived from three independent experiments. (H) CD81 suppresses Rac1 activity at cell–cell contacts during cell collisions. Quantification of FRET ratios in Ctrl MCF7 (Ctrl KD) and CD81 KD MCF7 cells (CD81 KD) collided with wildtype MCF7 (WT) cells. Rac1 activity at cell–cell contacts relative to cell-free edges is depicted as the ratio of FRET signals (mean grey values) at cell–cell contacts to the FRET signals (mean grey values) at the cell-free edge of the Raichu-Rac1-expressing cell (n = 71 for each condition, three independent experiments. All statistical analyses shown in the figure were performed using unpaired Student’s t test. Data are presented as arithmetic means ± SD; NS, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Source data are available for this figure: SourceData F8.

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