GEF-H1 and RhoA activate ERMs downstream of nocodazole-induced microtubule disassembly. (A and B) Rhotekin-RBD pull-down assays were performed to evaluate RhoA activation. Immunoblot of whole extract HEK293T cells incubated with vehicle (DMSO) or 1 µM nocodazole for 15 min are shown in the three top panels. Pull-down RhoA is shown in the lower panel (A). RhoA-GTP over total RhoA were quantified and normalized to vehicle (B). (C and D) Immunoblot of HEK293T cells pre-incubated with vehicle (DMSO), 1 µg/ml C3 transferase for 6 h or 10 µM Y-27632 for 4 h and then incubated with vehicle (DMSO) or 1 µM nocodazole for 15 min (C). p-ERM over ERM signals were quantified and normalized to vehicle (D). (E and F) Immunoblot of HEK293T cells stably expressing non-target shRNA (NT) or two different shRNA targeting GEF-H1 and incubated with vehicle (DMSO) or 1 µM nocodazole for 15 min. White lines indicate that intervening lanes have been spliced out (E). p-ERM over ERM signals were quantified and normalized to NT treated with vehicle (F). Immunoblots (A, C, and E) are representative of at least three independent experiments. p-ERM quantifications represent the mean ± SD of at least three independent experiments. Dots represent independent experiments (B, D, and F). P values were calculated using one-sample t test (B) or using Holm-Sidak’s multiple comparisons test with a single pooled variance (D and F) except for comparison made with normalized condition (vehicle + vehicle [D] or NT + vehicle [F]) where one-sample t test was applied. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Numbers associated with Western blots indicate molecular weight in kD. Source data are available for this figure: SourceData F2.