Fig. 2.
OPN interacts with CD44, and neutralization of CD44 inhibits OPN-stimulated apoptosis. ARVMs were treated with OPN (20 nM) for 24 h. Cell lysates were immunoprecipitated with anti-CD44 or anti-β1-integrin antibodies. Immunoprecipitates were analyzed by Western blot using anti-OPN (A and B) antibodies. Total ARVMs lysate treated with OPN served as positive (+ve) control. C: ARVMs were treated with OPN for 24 h. The cells were then used for proximity ligation assay (PLA) using anti-OPN and/or anti-CD44 antibodies. Middle: (−ve) PLA-staining using single (anti-CD44) antibody in OPN-treated cells and serves as a negative control. Fluorescent red staining in OPN-treated sample indicates interaction of OPN and CD44. D: ARVMs were pretreated with neutralizing anti-CD44 (IM7) or control IgG for 30 min followed by treatment with OPN for 24 h. Apoptosis was measured using TUNEL assay. *P < 0.05 vs. control (CTL); $P < 0.05 vs. OPN; #P < 0.05 vs. IM7 + OPN; n = 6.