Journal: Molecular cancer

Article Title: Differential G protein subunit expression by prostate cancer cells and their interaction with CXCR5.

doi: 10.1186/1476-4598-12-64

Figure Lengend Snippet: Figure 2 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.

Article Snippet: LNCaP, C4-2B, and PC3 cells were transfected with 1 μg control siRNA, Gαq/i2 siRNA, or Gα13 siRNA duplex (Santa Cruz, CA, USA) prior to harvest, and added to the top chambers in serum-free RPMI medium at 10,000 cells per well.

Techniques: Expressing, SDS Page, Western Blot, Immunoprecipitation, Staining, Control

Journal: Molecular cancer

Article Title: Differential G protein subunit expression by prostate cancer cells and their interaction with CXCR5.

doi: 10.1186/1476-4598-12-64

Figure Lengend Snippet: Figure 3 Validation of Gαq/11, Gαi2, and Gα13 protein association with CXCR5. Cell lines were treated with or without CXCL13 and lysed. (A) Gαq/11 and (B) Gαi2 were immunoprecipitated (IP) from total cell lysates. The IP cell lysates were resolved by SDS-PAGE and CXCR5 expression was examined by immunoblot. (C) Identification of CXCR4 and CXCR5 coupled to Gα13 following CXCL13 stimulation. 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 CXCR5 followed by CXCR4, after stripping. In all the experiments, GAPDH served as loading control. All experiments were repeated three times with unvarying results.

Article Snippet: LNCaP, C4-2B, and PC3 cells were transfected with 1 μg control siRNA, Gαq/i2 siRNA, or Gα13 siRNA duplex (Santa Cruz, CA, USA) prior to harvest, and added to the top chambers in serum-free RPMI medium at 10,000 cells per well.

Techniques: Biomarker Discovery, Immunoprecipitation, SDS Page, Expressing, Western Blot, Stripping Membranes, Control