Western Blot Analysis of Cleaved Notch1, c-Myc, p19Arf, and p-p53

Proteins were extracted from cells or tissues using RIPA lysis buffer (Sigma-Aldrich). The protein concentration was determined using a BCA protein assay kit (Thermo Scientific). A total of 30 mg of total protein were loaded for electrophoresis into 4–20% sodium dodecyl sulfate polyacrylamide gels (Bio-Rad). Separated proteins were transferred to a PVDF membrane (Bio-Rad). Membranes were blocked with 5% BSA in TBS with 0.1% Tween 20. Protein levels were detected using antibodies against cleaved Notch1 (Val1744) (Cell signaling Technology #2421, 1 to 1000 dilution)^{52 (link)}, c-Myc (Cell signaling Technology #2272S, 1 to 1000 dilution)^{53 (link)}, p19Arf (Novus Biologicals, clone: 5-C3-1, 1 to 500 dilution)^{54 (link)}, p-p53 (S15) (Cell signaling Technology #9284S, 1 to 500 dilution)^{55 (link)} and actin (BD Biosciences, 1 to 5000 dilution) followed by secondary horseradish-peroxidase conjugated antibodies (GE Health Care Life Sciences, 1 to 5000 dilution). Bands were visualized using ECL Plus western blotting detection reagents (Thermo Scientific). Uncropped scans of Western blots can be found in Supplementary Fig. 8.