Rhoa activation assay kit

G418, Fetal bovine serum (FBS), phallotoxins, RPMI 1640, DMEM, non-essential amino acids (NEAA), subcellular protein fractionation kit for cultured cells and Lipofectamine^® 2000 Transfection Reagent were from Thermo Fisher (Waltham, MA, United States). Transwell^® polycarbonate membrane cell culture inserts were bought from Corning (Tewksbury, MA, United States). Anti-vimentin (ab8978), -CCNY (ab107012), -snail (ab167615), -PFTK1 (ab224098), -β-catenin (ab6302), -TPM4 (ab181156), and -ZEB1 (ab124512) antibodies were purchased from Abcam (Cambridge, United Kingdom). The Rac1/Cdc42 activation assay kit and RhoA activation assay kit were obtained from Millipore (Billerica, MA, United States). Hoechst 33342 were obtained from Sigma (St. Louis, MO, United States). Matrigel Matrix was purchased from BD Science (San Diego, CA, United States).

To determine the activation of RhoA and Rac1 in FLSs of RA, OA, and patients of knee trauma, and the effect of Smo on RhoA and Rac1 activation in RA-FLSs, pull-down assays were performed according to the manufacturer’s protocol (RhoA activation assay kit and Rac1 activation assay kit, Millipore, MA, USA). Briefly, at 80% confluency, FLSs were lysed and the lysates were collected and stored at −80°C for the pull-down assay. Thirty microliters of the Rho or Rac1 Assay Reagent were added to 500 μl cell extract and the reaction mixtures were incubated for 45 min at 4°C with gentle agitation. After brief centrifugation (10 s, 14,000 × g, 4°C), the agarose beads were washed three times with 1× MLB, the supernatant was removed, and the agarose beads were re-suspended in 2× Laemmili-reducing sample buffer. Bound proteins were collected and examined by Western Blot analysis as previously described. GTP-RhoA or GTP-Rac1 was detected using anti-RhoA (3 μg/ml, Millipore, MA, USA) or anti-Rac1 antibodies (1 μg/ml, Millipore, MA, USA), respectively.

A RhoA activity assay was performed and quantified using the RhoA activation assay kit according to the manufacturer's instructions (Millipore, Massachusetts, USA). Briefly, HMEC‐1 were incubated with dasatinib (100 ng/mL) solubilized in Tris‐buffered saline containing 0.25% deoxycholic acid, 1% NP‐40, 1 mmol/L EDTA, and a phosphatase/protease inhibitor cocktail mixture (Thermo Fisher Scientific, Waltham, MA, USA). At various time intervals, cells were lysed and the cell lysate was incubated with Rhotekin‐linked agarose beads at 4°C for 60 min. The beads were washed with wash buffer and denatured. Active RhoA (GTP‐bound Rho) was detected by western blotting with an anti‐RhoA antibody.