Rabbit anti egfr

Co‐immunoprecipitation (Co‐IP) experiments were performed using the Thermo Fisher Scientific Pierce Co‐IP Kit (26149), following the manufacturer's protocol. In brief, cells were exposed to drugs when cell confluency reached 60–70%. After 4 or 24 h of drug exposure, cells were lysed with an ice‐cold Lysis/Wash Buffer supplemented with fresh protease and phosphatase inhibitors (Roche). Cell lysates were precleared by incubating the lysate with Control Agarose Resin at 4 °C for 1 h. To prepare antibody‐immobilized AminoLink Plus Coupling Resin, 25 μL Coupling Resin, and 1 μg of rabbit anti‐EGFR (Santa Cruz Biotechnology, Dallas, TX, USA; sc‐120) or IgG control antibody (Invitrogen, Waltham, MA, USA; 02‐6102) were co‐incubated in a spin column for 2 h at room temperature. Columns were then washed twice with 1× Coupling Buffer and six times with Wash Solution, and centrifuged after each wash. Subsequently, precleared cell lysate was added to the column containing antibody‐coupled resin, and incubated overnight at 4 °C with gentle mixing. Proteins were eluted using 60 μL of Elution Buffer, and eluates were analyzed by western blotting with primary antibodies for EGFR, pEGFR, PI3Kβ, and tubulin. See Supplementary Information section for antibody details. Table S1 in the Supporting Information section lists the antibodies and dilutions used for Co‐IP.

Western blotting analysis was performed as previously described (23, 38). Primary antibodies for Western blotting analysis were rabbit polyclonal anti-GAPDH (Cell Signaling, San Jose, CA) and rabbit anti-EGFR (Santa Cruz Biotechnology). Secondary antibody is HRP-conjugated anti-rabbit (Dako, Carpinteria, CA).