Figure 5.

Group V secretory phospholipase A₂ (sPLA₂V) is co-localized with endothelial protein C receptor (EPCR) and blocks activated protein C (APC) binding to rheumatoid synovial fibroblasts (RASFs). (A) sPLA₂V was co-localized with EPCR in rheumatoid arthritis (RA) synovial tissue, detected by immunofluorescent staining. Nuclei were counterstained by 4′-6-diamidino-2-phenylindole (DAPI) (blue). Scale bar: 50 μm. (B) sPLA₂V and EPCR expression by MCF-7 cells (M, used as a positive control), RASFs (RA), or OASFs (OA), detected by immunoprecipitation using anti-EPCR antibody and followed by Western blot using anti-EPCR or sPLA₂V antibody under non-reducing conditions. Red arrow indicates EPCR and sPLA₂V complex. Black arrow indicates EPCR. (C) APC in whole cell lysates of RASFs transfected with small interfering RNA (siRNA) for control (Con) or sPLA₂V siRNA for 48 hours and treated with APC (10 μg/mL) for 24 hours and detected by Western blot. (D) APC in the whole cell lysates of RASFs treated with recombinant APC (2 μg/mL), recombinant sPLA₂V (2 μg/mL) for first 30 minutes then APC (sPLA₂V + APC) or APC for first 30 minutes then sPLA₂V (APC + sPLA₂V) for 4 hours, detected by Western blot. The images represent one of three different experiments using three different RASF cell lines. Data on (C) and (D) were semi-quantified by image analysis software in comparison with β-actin, expressed as a percentage of control and shown as mean ± standard deviation (SD) (n = 4) and analyzed by one-way analysis of variance followed by Tukey’s honestly significant difference (HSD) post hoc test. **P <0.01.