Anti rhoa sc 418

RhoA activity in cultured cells was assessed as described by Chikumi and colleagues [49 (link)]. Briefly, cells were grown to 70% confluence in 100 mm dishes, serum-starved for 18 hours and then stimulated with 1U/ml thrombin (Sigma, MO, USA) or 5 uM lysophosphatidic acid (LPA) (Sigma-Aldrich, MO, USA) for 30 seconds to 10 minutes. Cells were lysed on ice with 600 μl of cold lysis buffer (detailed above). Lysates were then collected and centrifuged at 14000 rpm for 5 minutes at 4°C. From the resulting supernatants, 100 μl was used for total RhoA protein levels, while the remaining 500 μl for each sample were incubated with glutathione-Sepharose 4B beads (GE Healthcare, Sweden) bound to glutathione-S-transferase (GST)-rhotekin-RhoA binding domain for 30 minutes at 4°C on a rotator. The beads were collected by centrifugation at 9000 rpm for 1 minute at 4°C and washed (x3) with cold lysis buffer. The associated GTP-bound RhoA was released with protein loading buffer and denatured by heating for SDS-polyacrylamide gel electrophoresis followed by western blotting. Blots were incubated using anti-RhoA (sc-418; Santa Cruz Biotechnology, CA, USA) at 1:1000 dilution for 1 hour and further processed according to western blotting conditions mentioned above.

Antibodies used in this study include anti-phospho-210 PLK1 (BD 558400), anti-γ-tubulin (Thermo MA1-20248, clone GTU-88), anti-α-tubulin (MAB1864 Millipore), anti-anillin (polyclonal rabbit, Kim and Burkard, unpublished), anti-Plk1 (F-8, Santa Cruz Biotechnology sc-17783), anti-β-actin (AC-15, ab6276), anti-flag (M2) HRP (Sigma A8592), anti-RhoA (SC418 Santa Cruz), anti-pericentrin (ab44448 Abcam), anti-ACA (HCT0100 Immunovision), and anti-mouse HRP (Jackson Immunoresearch Laboratories Inc. #115-035-003). Immunoprecipitation of flag constructs was performed using anti-flag M2 affinity gel (Sigma A2220). For immunofluorescence, Alexa-flour antibodies were used (Invitrogen). Mitotic index was determined through Hoechst 33258 staining and microscopy. Crystal violet stain is composed of crystal violet (Sigma C-0775) with buffered formalin (Sigma HT-50-1-128)