Semi dry transfer blotter

General electrophoresis, horizontal and vertical electrophoresis tank, and semidry transfer blotter were purchased from Bio-Rad Laboratories Inc., (Hercules, CA, USA). Inverted fluorescence microscope was purchased from Olympus (CK53, Japan). A microplate reader was purchased from Tecan Group Ltd. (Männedorf, Switzerland); and the automatic developing instrument (chemo doc) was purchased from BioRAD.
Remove the wax block of the gastric cancer patient and the paraffin block from the pathology department and send them to the partner company for testing after taking the paraffin slice.

Protein lysates were obtained from HORCs using Mammalian Protein Extract Reagent M-PER supplemented with Halt Phosphatase Inhibitor Cocktail, Protease Inhibitor Cocktail and 5mM EDTA (All from Thermo Scientific, Loughborough, UK) for 20min on ice followed by centrifugation at 13,000rpm for 5min. Protein concentration of each lysate was determined using a bicinchoninic acid (BCA) protein assay (Thermo Scientific, Loughborough, UK). Equal amounts of protein were loaded onto 10% SDS-PAGE gels and proteins separated by electrophoresis. Proteins were transferred to PVDF membrane (Perkin Elmer Life Sciences, Cambridge, UK) using a semi-dry transfer blotter (Bio-Rad Laboratories, Hemel Hempstead, UK). Membranes were blocked with PBS-T (0.1% Tween-20 in PBS, 5% fat-reduced milk), hybridized with primary antibody followed by incubation with secondary antibody (GE Healthcare, Buckinghamshire, UK). Bands were visualised using chemiluminescent ECL Plus Western Blot Detection reagent (GE Healthcare, Buckinghamshire, UK) and net band intensity determined (1D 3.5 software, Eastman Kodak, Rochester, NY). Primary antibodies (Cell Signaling Technology, Danvers, MA, USA) against phospho- and total p38, phospho- and total JNK were used at 1:250, 1:1000, 1:500 and 1:500 respectively.