Fig. 2.

Cadherin-11 cleavage stimulation by ionomycin. (a) Proposed cadherin-11 cleavage model shows that cadherin-11 is first cleaved extracellularly at the plasma membrane by a cell sheddase, generating a C-terminal fragment 1 (CTF1) and an extracellular domain fragment (sCad11). Then CTF1 is cleaved near the intracellular plasma membrane, releasing C-terminal fragment 2 (CTF2) into the cytosol. CTF2 is likely rapidly degraded by the proteasome but may also effect gene transcription. (b) Lysates from RA synovial fibroblasts stimulated with 5 μM ionomycin as indicated were analyzed by western blot using antibodies directed against cadherin-11 extracellular (3H10) or intracellular (5B2H5) epitopes (FL, full length cadherin-11). (c) Surface staining for cadherin-11 (3H10), MHC class I (W632) or isotype control on RA synovial fibroblasts before or after 1 hour ionomycin stimulation was determined by flow cytometry (representative, 2 experiments). (d) Culture media and cell lysates harvested from RA synovial fibroblasts treated with or without ionomycin for 1 hour were analyzed by western blot for extracellular (3H10) and intracellular (5B2H5) cadherin-11 epitopes. Culture media was left unconcentrated or concentrated approximately 12-fold (representative 3 experiments). (e) Culture media from unstimulated cells was immunoprecipitated with anti-cadherin-11 antibody 23C6 or appropriate isotype control prior to western blot analysis (representative 3 experiments). (f) CTF1 levels in twelve sequential experiments were assayed by measuring band pixel intensity inunstimulated and ionomycin-stimulated cell lysates by western blot using standard and longer exposure times. (Pooled, 6 cells lines. Statistical comparison to background by t-test. n.s. = not significantly different; * p<0.001).