Pull-Down Assay

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.