Figure 7. pERMs negatively regulate... | Rockefeller University Press

$pERMs negatively regulate myosin activation through RhoA.(A) Jeg-3 wild-type and knockout cells were either treated with DMSO, 10 nM calyculin A for 10 min, or 1 µM Y-27632 for 30 min or cotreated with 10 nM calyculin A and 1 µM Y-27632 before cell lysate extraction. Extracts were then blotted with pMLC (T18/S19) and total MLC antibodies. (B) Quantification of pMLC staining normalized to MLC as a loading control. Bars show mean ± SEM; n = 7 for DMSO and n = 4 for other drug conditions. P values were calculated against wild type using the t test (*, P ≤ 0.05). (C) Jeg-3 wild-type and knockout cells treated with either DMSO, 10 nM calyculin A (CalA) for 10 min, or 1 µM Y-27632 for 30 min or cotreated with 10 nM CalA and 1 µM Y-27632 before fixation and staining with pMLC and actin. Images are max Z-projections of apical cross-sections. (D) Representative Western blot of active RhoA-GTP pull-down results in Jeg-3 cells. Upper row: RhoA blot of the active fraction of RhoA (GTP) pulled down by rhotekin beads. Lower row: Blot of RhoA of 5% of input lysate. (E) Quantification of pull-down represented in D. Mean ± SEM; n = 10 for no treatment and n = 5 for Y-27632 (t test; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (F) LOK-CTD-GFP-Flag pull-down of cells expressing constitutively active RhoA (RhoA-L30), indicating that C-terminal LOK binds active RhoA. WB, Western blot. (G) Model of RhoA signaling negatively regulated by pERMs (dotted blue line).$

Figure 7.

pERMs negatively regulate myosin activation through RhoA. (A) Jeg-3 wild-type and knockout cells were either treated with DMSO, 10 nM calyculin A for 10 min, or 1 µM Y-27632 for 30 min or cotreated with 10 nM calyculin A and 1 µM Y-27632 before cell lysate extraction. Extracts were then blotted with pMLC (T18/S19) and total MLC antibodies. (B) Quantification of pMLC staining normalized to MLC as a loading control. Bars show mean ± SEM; n = 7 for DMSO and n = 4 for other drug conditions. P values were calculated against wild type using the t test (*, P ≤ 0.05). (C) Jeg-3 wild-type and knockout cells treated with either DMSO, 10 nM calyculin A (CalA) for 10 min, or 1 µM Y-27632 for 30 min or cotreated with 10 nM CalA and 1 µM Y-27632 before fixation and staining with pMLC and actin. Images are max Z-projections of apical cross-sections. (D) Representative Western blot of active RhoA-GTP pull-down results in Jeg-3 cells. Upper row: RhoA blot of the active fraction of RhoA (GTP) pulled down by rhotekin beads. Lower row: Blot of RhoA of 5% of input lysate. (E) Quantification of pull-down represented in D. Mean ± SEM; n = 10 for no treatment and n = 5 for Y-27632 (t test; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (F) LOK-CTD-GFP-Flag pull-down of cells expressing constitutively active RhoA (RhoA-L30), indicating that C-terminal LOK binds active RhoA. WB, Western blot. (G) Model of RhoA signaling negatively regulated by pERMs (dotted blue line).

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