Tris hcl denaturing polyacrylamide ready gel

TKE2 or HEK293 cells were homogenized in RIPA buffer (50mM Tris base, 150mM NaCl, 0.5% deoxycholic acid-sodium salt, 2% SDS, and 1% NP40, pH 7.5) containing 1x protease inhibitor cocktail (Sigma P8340). Cell lysates (20µg) from each sample were separated on a 4–20% linear gradient Tris-HCl denaturing polyacrylamide Ready Gel (Bio-Rad) and transferred to PVDF membrane (Whatman). Membranes were blocked with 5% nonfat milk in TBST (10mM Tris-HCl pH 8.0, 150mM NaCl, 0.05% Tween 20) and probed with primary antibody in the same buffer overnight at 4°C. After three washes in TBST, membranes were probed with HRP-conjugated secondary antibody for an hour at room temperature and bond second antibody was further detected using an enhanced chemiluminescence assay (Supersignal West Pico, #34080; Thermo-Fisher Scientific) and examined and photographed using a VersaDoc 4000MP imaging system (Bio-Rad). Primary antibodies, rabbit anti-Shp2 (D50F2) (cell signaling, Cat: 3397P), rabbit anti-∆Np63 (Abcam, ab166857), rabbit anti-Ngf (LSBio LS-C171793), and goat anti-β-actin (Santa Cruz, sc-1616) were used to examine expression of Shp2, ∆Np63, Ngf, and β-actin, respectively. The secondary antibodies, goat anti-rabbit IgG-HRP (Thermo-Fisher Scientific, G-21234), donkey anti-goat IgG-HRP (Thermo-Fisher Scientific, A16005) were used to assist in the detection by western blots.