Supplementary Figure 3: p190B RhoGAP degrades the junctional Rho zone when NMII is inactivated.

(a–e) ECT2 (a), MgcRacGAP (RacGAP) (b), p190A Rho GAP (p190A), Myosin IIA and Myosin IIB (c,e) localization in control (Ctrl), NMIIA RNAi (IIA KD), NMIIB RNAi (IIB KD), DMSO or blebbistatin (BLB, 100 μM, 2 h) treated MCF-7 cells. (f,g) Lysates from control (Ctrl), NMIIA RNAi (IIA KD), NMIIB RNAi (IIB KD), DMSO or blebbistatin (BLB, 100 μM, 2 h) treated MCF-7 cells immunoblotted for p190B Rho GAP and β-tubulin (loading control). (h) Rac 1 localization in control (Ctrl), NMIIA RNAi (IIA KD), NMIIB RNAi (IIB KD), DMSO or blebbistatin (BLB, 100 μM, 2 h) treated MCF-7 cells. (i) MCF-7 cells transfected with non-targeting control siRNA or Rac1 siRNA (Rac1KD) were treated with DMSO or with blebbistatin (BLB; 100 μM, 2 h), then fixed and stained for p190B Rho GAP. (j) Lysates from MCF-7 cells transfected with non-targeting control siRNA (Ctrl) or transfected with siRNA against Rac1 (Rac1KD) were immunoblotted for Rac1 and β-tubulin (loading control). (k) MCF-7 cells were treated with DMSO or blebbistatin (BLB; 100 μM, 2 h) alone or in combination with Rac inhibitors EHT1864 (10 μM, 12 h) or NSC23766 (50 μM, 12 h), fixed with methanol and stained for p190B GAP. (l) MCF-7 cells transduced with lentivirus encoding non-targeting control shRNA (Ctrl), NMIIA (IIAKD) or NMIIB (IIB KD) shRNA were transfected with the Raichu-Rac FRET biosensor and FRET measurements were performed as described in the Methods section. Average emission ratios were calculated at the apical junctions. Data represent mean ± s.e.m. of 30 contacts (n = 30) and the result is representative of two independent experiments. Statistical information, P values and source data are in Supplementary Table 2. (m) MCF-7 cells were treated with DMSO or treated with blebbistatin (BLB) (100 μM, 2 h) alone or in combination with Src inhibitors SU6656 (10 μM) or PP2 (10 μM)) for 4 h. Cells were fixed with methanol and stained for p190B GAP. Scale bars, 10 μm.