Anti phospho egfr sampler kit

HT29 cells were seeded in 12-well flat-bottom plates with 500 μl of serum-free medium for 16 h. EGF (#E9644, Sigma) was reconstituted to a concentration of 1 mg/ml using 10 mm acetic acid, diluted with 0.1% bovine serum albumin, and added to the HT29 cells at concentrations of 100 ng/ml for 10, 20, and 120 min in separate wells. In addition, QRH*-Cy5.5 at concentrations of 5 and 100 μm for 10, 20, and 120 min was added to separate wells. The cells were washed with PBS and lysed in RIPA buffer containing protease inhibitors (#11836170001, Roche, Basel, Switzerland). Lysates were separated by gel electrophoresis, transferred to polyvinylidene difluoride membranes (#ISEQ00010, Millipore), and detected by immunoblotting using an enhanced chemiluminescence system (#RPN2106, GE Healthcare, Little Chalfont, UK). Anti-EGFR antibody (#2232S, Cell Signaling Technology), anti-phospho-EGFR sampler kit (#9922s, Cell Signaling Technology), anti-AKT (#4691P, Cell Signaling Technology, Danvers, MA), anti-ERK1/2 (#4695P, Cell Signaling Technology), anti-phospho-AKT (pS473; #4060P, Cell Signaling Technology), anti-phospho-ERK1/2 (#4370P, Cell Signaling Technology), and anti-tubulin (#32–2600, Invitrogen) were used as per the manufacturer's instructions.