Rabbit anti tks5 sh3 1

siRNA targeting human Tks5, control siRNA, GST, and Myc (clone 9E10) antibodies, goat anti-Tks5 polyclonal antibody (M-20), and p21 antibody were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-Tks5 polyclonal antibody was a gift of S. Courtneidge (Sanford/Burnham Medical Research Institute, La Jolla, CA). XB130 monoclonal antibody was generated as previously described (Xu et al., 2007 (link)). XB130 siRNA and control siRNA were from GE Healthcare Dharmacon (Lafayette, CO). RGS-His antibody to detect 6xHis-tagged proteins was from Qiagen (Valencia, CA). Src (clone GD11) monoclonal antibody, phosphotyrosine (clone 4G10) monoclonal antibody, and GADPH antibody were from Upstate Biotechnology (Lake Placid, NY). Horseradish peroxidase–conjugated goat anti-mouse or anti-rabbit secondary antibodies were from Amersham Pharmacia Biotech (Piscataway, NJ). PI3K p85α, phosphotyrosine 458 PI3K, Akt, and phosphoserine 473 Akt antibodies were from Cell Signaling Technology (Beverly, MA). Anti-GAPDH antibodies were from Abcam (Cambridge, United Kingdom). Human Ki67 antibody and rabbit anti-Tks5 (SH3#1) were from EMD Millipore (Merck, Darmstadt, Germany).

Cells were lysed with RIPA buffer for Western blot analysis or Triton X-100 lysis buffer for immunoprecipitation procedures. Protein samples were analyzed using the Pierce 660-nm protein assay (Thermo Scientific, Rockford, IL), and an initial protein concentration of 250 μg/ml of the whole-cell lysate was used as the input for immunoprecipitation reactions. Equal volumes were used for the reactions. The whole-cell lysate was incubated with primary antibody against each protein (XB130 monoclonal antibody was generated as previously described; Xu et al., 2007 (link)), rabbit anti-Tks5 (SH3#1) from EMD Millipore, and Src (clone GD11) monoclonal antibody from Upstate Biotechnology, washed with Triton X-100 lysis buffer (1.0% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl, pH 8.0), and eluted by boiling for 10 min at 50°C with 50 μl of 2× SDS Laemmli buffer (no dithiothreitol), and 15 μl of sample was loaded on each lane. Coimmunoprecipitation used protein G–Sepharose beads precleared with normal mouse IgG. Rabbit IgG and mouse IgG2a and IgG2b were used as controls for Tks5 and XB130 immunoprecipitation. SuperSignal Dura Chemiluminescent substrate (Thermo Scientific, Rockford, IL) was used to detect protein signal after transfer.