Fig. 3.

Stimulation of cadherin-11 cleavage by TNF-α. (a) Lysates from rheumatoid arthritis (RA) synovial fibroblasts left untreated or treated with ionomycin, TNF-α, or platelet-derived growth factor (PDGF)-BB for 2 hours were assayed for cadherin-11 cleavage using monoclonal antibody 5B2H5. (b) Mean fold increase in C-terminal fragment 1 (CTF1) pixel intensity for the indicated stimulation over unstimulated controls was calculated across several experiments using RA fibroblasts from different donors (fold change over media control (mean +/− standard error of mean): ionomycin (n = 7, 4 cell lines) 4.6 +/− 2.1; TNF-α 25 ng/ml (n = 74 cell lines) 1.7 +/− 0.17; PDGF-BB 100 ng/ml (n = 53 cell lines) 0.89 +/− 0.074). (c) Synovial fibroblast micromasses were cultured for 21 days, transferred to media containing 0.2 % serum for 24 hours, and then stimulated for 5 days with or without TNF-α (10 ng/ml). Cadherin-11 cleavage was then assessed using a rabbit polyclonal antibody against the cadherin-11 cytoplasmic domain (representative of at least three separate experiments with two cell lines). Equal protein loading was confirmed by β-actin staining