association with PAR-1 and CXCR5, and G
contribution to PCa cell lines invasion and Rac/Rho activation. (A) Cell lines were treated with or without CXCL13 and lysed. Antibody against Gα13 was used to immunoprecipitate (IP) it from total cell lysates. The IP cell lysates were resolved by SDS PAGE and immunoblotted for PAR-1 followed by CXCR5, after stripping. GAPDH served as a loading control. (B) Invasion of LNCaP, C4-2B and PC3 cells was assessed using BD Matrigel™ invasion chamber. The assay was performed using LNCaP (open bars), C4-2B (solid bars) and PC3 (hashed bars) cells transfected and/or treated with control siRNA, Gαq/αi2 siRNA, or Gα13 siRNA duplex, and CXCL13 and/or Thrombin for 8 h and the cells that migrated to the lower surface of the membrane were counted by microscopy at 40X magnification. CXCL13-treated cells exhibited an enhanced ability to invade Matrigel. Abrogation of Gαq/i2 decreased the ability of cells to invade whereas silencing of Gα13 did not affect cell invasion. (C) Rac and RhoA protein expression were determined in CXCL13 and/or thrombin treated LNCaP, C4-2B, and PC3 cells. (i) shows differential expression of Rac protein, involved in lamellipodia formation, in response to CXCL13, thrombin, CXCL13 followed by thrombin and from cells transfected with Gαq/i2/13 siRNA in different experiments. (ii) shows differential expression of RhoA protein, involved in stress fiber formation and cell adhesion, in response to CXCL13, thrombin, CXCL13 followed by thrombin and from cells transfected with Gα13 siRNA in different experiments. All experiments were repeated at least three times and results were in accordance with each other.