Horseradish peroxidase hrp labeled secondary antibodies

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.

Protein lysates were prepared with radio immunoprecipitation assay (RIPA) buffer. Protein lysates were separated utilizing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), and were incubated with antibodies listed in Supplementary Table 1 followed by horseradish peroxidase (HRP)-labeled secondary antibodies (Bio-Rad, USA). For individual studies, the densitometry of individual blot signals from three independent western blot experiments were quantified using Image J software. The individual values for each blot signal was normalized to respective controls followed by the statistical analysis among different samples (Student’s t-test).