Figure 4

RasGRF1 is expressed as a truncated protein in fibroblast-like synoviocytes. (a) Immunoblot analysis of Ras guanine nucleotide-releasing factor 1 (RasGRF1) in rheumatoid arthritis (RA) and osteoarthritis (OA) fibroblast-like synoviocytes (FLS). The 140 kDa, 98 kDa, 75 kDa and 54 kDa proteins reacting with RasGRF1 antibodies are indicated on the left by arrowheads. Relative mobility of molecular weight (Mw) standards (kDa) indicated to the right. (b) Expression of 140 kDa, 98 kDa, 75 kDa, and 54 kDa RasGRF1 polypeptides as well as the total RasGRF1 signal, normalized to tubulin expression, was quantified in RA (n = 5) and OA (n = 5) FLS lines, and expressed as mean optical density ± standard error of the mean (SEM). (c) Overexpression of RasGRF1 in RA FLS. RA FLS were treated with transfection reagent alone (mock) or transfected with empty (control) vector or vector encoding RasGRF1, and cell lysates immunoblotted with antibodies against RasGRF1 (upper panel) and tubulin (lower panel). Expression of full-length and truncated RasGRF1 polypeptides is indicated with arrows, and a 60 kDa polypeptide with an asterisk. (d) Expression of 140 kDa, 98 kDa, 75 kDa, and 54 kDa RasGRF1 polypeptides following transfection of RA FLS with empty vector or RasGRF1, normalized to tubulin expression was quantified and expressed as mean optical density ± SEM (middle panel) (n = 4). (e) Silencing of RasGRF1 expression with locked nucleic acid (LNA). RA FLS were treated with transfection reagent alone (mock) or transduced with control or RasGRF1 LNA and lysates assessed for expression of RasGRF1 (upper panel) and tubulin (lower panel) by immunoblotting. (f) Quantitative analysis of (e) as in (d). *P < 0.05, **P < 0.01 compared with controls.