Anti phospho egfr antibody

Protein samples were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA). Membranes were blocked with fat‐free milk combined with tris‐buffered saline plus tween 20 for one hour at room temperature and then incubated with the appropriate primary antibody and horseradish peroxidase conjugated secondary antibodies. Imager was used to visualize the blots. The primary antibodies used in this study were as follows: anti‐CDK4 (#12790, 1:1000, cell signaling), Anti‐Cyclin D1 antibody (#2978, 1:1000, cell signaling), Anti‐phospho‐EGFR antibody (#47724, 1:1000, cell signaling), Anti‐Phospho‐RB (ser780, #9307, 1:1000, cell signaling), anti‐E2F1 (#666515,1:1000,Proteintech), CDKN2a (#80772, 1:1000, cell signaling); and β‐actin (ab92552, 1:1000).

MKN45 and MKN74 cells were lysed and proteins (50 μg) were separated by 10% SDS- polyacrylamide gels, followed by electro-transfer onto a nitrocellulose membrane. For EGFR and Erk1/2 western blotting only MKN45 cells were lysed and proteins (50 μg) were separated by 8% and 12% SDS-polyacrylamide gels respectively, followed by electro-transfer onto a nitrocellulose membrane. The membranes were sequentially incubated with blocking buffer (TBS-Tween containing 5% nonfat dry milk or 5% BSA respectively) for 1 hour at room temperature, and then overnight at 4°C with one of the following antibodies: anti-TGR5 (1:2000, #ab72608 Abcam), anti-FXR (1:2000 #sc-13063 Santa Cruz Biotechnology), anti-EGFR (1:1000, #2232 Cell Signaling), and anti-phospho-EGFR antibody (1:1000, #4407 Cell Signaling), anti-Erk1/2 (P44/42 MAPK) (1:1000, #46955 Cell Signaling), anti-phospho-Erk1/2 (p-P44/42 MAPK) (1:1000, #91015 Cell Signaling). Primary antibody were detected with the horseradish peroxidase (HRP)-labeled secondary antibodies (1:10000, BioRad) for 1 hour at room temperature. After washing with TBST, protein bands were visualized by Lite Ablot TURBO (Euroclone) according to the manufacturer's instructions.