![Figure 1. Involvement of tyrosine dephosphorylation in ROCK-dependent cell rounding. (A–C) Adherent D2 cells plated in culture dishes in serum-free medium for 2 h were serum stimulated and treated with 300 μM orthovanadate (Na3VO4) or 20 μM Y27632, followed by stimulation with 20% heat-inactivated serum for 10 min. (A) Phase-contrast images of cells with indicated treatment. (B) Western blots of cells probed for phosphorylated[T18/S19] and total MLC. (C) Endogenous RhoA activity measured by GST-RBD pulldown assay as described in Materials and methods. Values are mean ± SD of three independent experiments. (D and E) Cells were plated on FN-coated dishes for 2 h in serum-containing medium treated with 20 μM Y27632 or 300 μM orthovanadate, followed by addition of 20 μg/ml of TAT-RhoAV14 for 30 min. (D) Phase-contrast images of cells. (E) Western blot of cells probed by antibodies against phospho-MLC, total MLC, and HA for TAT-RhoAV14. Bars, 50 μm.](https://cdn.rupress.org/rup/content_public/journal/jcb/181/6/10.1083_jcb.200710187/6/jcb_200710187_gs_fig1.jpeg?Expires=1773607121&Signature=Jr8jU8dJuK-iUwKhOKf~do~c9tQv1omrKkcPeZyRtZZHhZXevQ0ufdMs-NfBZ6X-pf2u5v1WEoO6opyJF4lEn34u0zGqtRYOamcQ4kjC2hucyIuKNuX6WACEM2alA3seBqy72eId6ihv1x6n1tiEJ~EPFhDs8HssXpAcoGLFWZxLnEDuVNrEWtVNuZXq3UGgEvV1EPmc1QVbz8KHBM3HmXiYL~5cI8Adth5lzTxfWG4-GWCB9HCCUSkmRltLA-1lr5dR-xkv5VfTqBHr~HOJbYzE4TIShSRSlDSpyx~MMsFpqKLmrJOVqlZG8WY29qhCxJ3gOCoV5hCpnk3KgNoBAA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Involvement of tyrosine dephosphorylation in ROCK-dependent cell rounding. (A–C) Adherent D2 cells plated in culture dishes in serum-free medium for 2 h were serum stimulated and treated with 300 μM orthovanadate (Na3VO4) or 20 μM Y27632, followed by stimulation with 20% heat-inactivated serum for 10 min. (A) Phase-contrast images of cells with indicated treatment. (B) Western blots of cells probed for phosphorylated[T18/S19] and total MLC. (C) Endogenous RhoA activity measured by GST-RBD pulldown assay as described in Materials and methods. Values are mean ± SD of three independent experiments. (D and E) Cells were plated on FN-coated dishes for 2 h in serum-containing medium treated with 20 μM Y27632 or 300 μM orthovanadate, followed by addition of 20 μg/ml of TAT-RhoAV14 for 30 min. (D) Phase-contrast images of cells. (E) Western blot of cells probed by antibodies against phospho-MLC, total MLC, and HA for TAT-RhoAV14. Bars, 50 μm.
