Figure 6. Determination of the role of CaR-mediated signaling in the cleavage of filamin A.

(A) DU145 and PC-3 cells were transfected with negative control (Neg) or CaR-specific siRNA (CaR), (B) DU145 and PC-3 cells were serum-deprived overnight last hour with different chemical regulators, (C and D) DU145 or PC-3 cells as well as their stable variants with silenced p115RhoGEF expression were serum deprived overnight. The serum deprived cells were treated with or without 3 mM CaCl₂ for 60 mins, and then the samples were processed for immunoblotting using the antibodies against CaR, p115RhoGEF, filamin A or tubulin. (E) DU145, PC3 and their stable variants were seeded in 24-well plates with Transwell filters, and then analyzed for Ca_o²⁺-induced cell migration. The data represent means of 40 fields with duplicate samples in two experiments. *P < 0.05. Con, control; CaR, Ca²⁺-sensing receptor; FL, full length of filamin A; 180 Fr, 180 kD fragment of filamin A; Tub, tubulin; Tetra, tetrandrine: FPL, FPL-64176; SKF, SK&F96365; and p115, p115RhoGEF. The clonal cells with stably silenced p115RhoGEF are from DU145 (C10 and C17) and PC-3 (C4 and C6) cells.