Western Blot Analysis of Cell Signaling Proteins

Total cell extracts were prepared in radioimmunoprecipitation assay buffer (20 mM Tris-HCL, pH 8.0, 137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 10% glycerol, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate), containing a protease inhibitor cocktail (Sigma-Aldrich, P2714), and incubated at 4°C for 1 h. Western blotting was performed as previously described (Perez-Garcia et al., 2021 (link)). In brief, protein lysates were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membrane (Immobilon-P, Millipore). Membranes were blocked with 5% milk powder and incubated with primary antibody overnight at 4°C, followed by horseradish peroxidise (HRP)-conjugated secondary antibodies. Blots were probed with the following antibodies: anti-ITGA2 (1:1000, Abcam, ab181548), anti-JAG2 (1:1000, Cell Signaling Technology, 2210), anti-Cleaved NOTCH1 (1:1000, Cell Signaling Technology, 4147), anti-NOTCH1 (1:1000, Cell Signaling Technology, 3608), anti-TP63 (1:1000, Abcam, ab124762), anti-IRF7 (1:1000, Cell Signaling Technology, 4920) and anti-β actin (1:5000, Abcam, ab6276). Horseradish peroxidase-conjugated secondary antibodies (used at a dilution of 1:3000) were sourced from Bio-Rad. Detection was carried out with enhanced chemiluminescence reaction (GE Healthcare, RPN2209) using standard X-ray films.