Expression of CXCR4, CXCR5 and associated G proteins in PCa cell lines. (A) Equal protein amounts (50 μg) from RWPE-1, LNCaP, C4-2B, and PC3 cell lysates were resolved by SDS-PAGE and the expression of CXCR5 (37 kDa) was determined by immunoblot. (B) Western blot analysis of CXCR4 expression with and without CXCL13 treatment. (C) & (D) Cell lines were treated with or without CXCL13 and lysed. CXCR5 was immunoprecipitated (IP) to pull down associated proteins from total cell lysates. The IP cell lysates were resolved by SDS-PAGE and the expression of (C) Gαi1, Gαi2, Gαi3, Gαs, Gαq/11, Gα12, Gα13 (D) Gβ1, Gβ2, Gβ3, Gβ4, and Gγ5, Gγ7, Gγ9, Gγ10 were examined by immunoblot. The blots in panel C and D were stripped each time and re-probed to stain different Gα, Gβ, and Gγ protein subunits. In all the experiments, GAPDH served as loading control. All experiments were repeated three times with unvarying results.