Arhgap5

Whole cell extracts were extracted using RIPA and protein concentrations were determined by BCA assay. Equal aliquots of 50 μg total proteins were separated by SDS‐PAGE. Then proteins were transferred to a PVDF membrane. Membranes were blocked with 5% bovine serum albumin in TBST and then incubated in primary antibodies overnight at 4°C followed by secondary antibodies for 1 h at 37°C. Rabbit polyclonal primary antibody against CXCR4 (sc‐9046), mouse monoclonal antibodies against RhoA (sc‐418), RhoGEF (sc‐166301), ROCK (sc‐17794), PI3K (sc‐166365), PAK (sc‐166174), PKN (sc‐136037), GAPDH (sc‐166545), and the horseradish peroxidase‐conjugated secondary antibodies against rabbit‐IgG (sc‐2004) and mouse‐IgG (sc‐2005) were purchased from Santa Cruz Biotechnology. The primary rabbit polyclonal antibody against ARHGAP5 was purchased from Sigma‐Aldrich. Finally, signal was visualized through a chemiluminescent detection system (Pierce ECL Substrate Western blot detection system, Thermo, Rockford, IL, USA).

The plasmid (NM_003467) for the construction of the human CXCR4 vector, which was also used in unmodified form as the negative control vector, was purchased from Genechem. The siRNA-Gα₁₃ was used to target human Gα₁₃, and the non-silencing siRNA (si-control; GenePharma) served as the control. AMD3100 (GenePharma), used to inhibit CXCR4, was dissolved in sterile PBS as a 1mg/ml stock solution and then diluted into the culture medium to a final concentration of 10, 100 or 1000 ng/ml. Rabbit polyclonal primary antibodies against CXCR4 (sc-9046), Gα₁₂ (sc-409), and Gα₁₃ (sc-410), mouse monoclonal antibodies against GAPDH (sc-166545), RhoA (sc-418), PAK (sc-166174), PKN (sc-136037), PI3K (sc-166365), ROCK (sc-17794), and RhoGEF (sc-166301), and the horseradish peroxidase–conjugated secondary antibodies against rabbit-IgG (sc-2004) and mouse-IgG (sc-2005) were purchased from Santa Cruz Biotechnology. The mouse monoclonal antibody against GAPDH (AB-2302) was purchased from Millipore (MA). The primary rabbit polyclonal antibody against ARHGAP5 was purchased from Sigma-Aldrich.