Fig. 4

Involvement of γ-secretase in cadherin-11 cleavage. (a) Cell lysates from synovial fibroblasts incubated overnight increasing concentrations of the γ-secretase inhibitor L-685-458 and then stimulated as indicated were analyzed for cadherin-11 cleavage (representative 5 experiments with 4 cells lines, full-length cadherin-11 (FL); cadherin-11 (Cad11)). (b) CTF1 nean pixel intensity was calculated in lysates from ionomycin-stimulated synovial fibroblasts pretreated with or without L-685-458 (dose range 300–3000 nM, * p=0.0304 by paired t-test, error bars reflect standard error of the mean). (c) Cell lysates from RA synovial fibroblasts incubated overnight with increasing concentrations of the proteosome inhibitor lactacystin and then stimulated as indicated were analyzed for cadherin-11 cleavage (representative of 9 experiments with 5 cells lines). (d) CTF2 mean pixel intensity was calculated in lysates from synovial fibroblast treated with or without ionomycin (5 μM) and lactacystin (3–10 μM) as indicated (p<0.0001 by one-way ANOVA, error bars reflect standard error of the mean). (e) RA synovial fibroblasts were transfected with the indicated siRNAs, incubated overnight with 10 μM lactacystin, and then were analyzed for cadherin-11 cleavage in the presence or absence of ionomycin (representative 3 experiments with 3 cell lines). Silencing was confirmed by western blot. β-actin levels were used fro protein loading control. (f) Mean pixel intensity was calculated for CTF2 bands in siRNA-transfected RA synovial fibroblasts pretreated with 10 μM lactacystin and then stimulated with ionomycin (* p=0.0045, paired t-test; **p=0.0136 paired t-test, n=3, error bars reflect standard deviation of the mean).