Western Blotting for Protein Expression

Protein extracts were prepared according to previously published protocol (27 (link)). Briefly 5 μL of protein lysates were loaded onto SDS-polyacrylamide gels and transferred to Immun-Blot PVDF membranes (Bio-Rad Laboratories). After blocking in 5% milk, the membranes were incubated in primary antibodies against the following: p63 (4A4, 1:20,000), ΔNp63 (E6Q3O; Cell Signaling Technology, 1:5000), ITGB1 (Proteintech, 1:10,000), ITGB4 (Proteintech, 1:10,000), cMYC (Santa Cruz Biotechnology, 1:5000), AKT1 (Proteintech, 1:10,000), mTOR (Proteintech, 1:10,000), Raptor (Proteintech, 1:10,000), S6 (Cell Signaling Technology, 1:5000), and p-S6 (Cell Signaling Technology, 1:5000). The MAB374 antibody (EMD Millipore) was used to detect GAPDH as a loading control at 1:20,000 dilution. HRP-conjugated secondary antibodies corresponding to the primary antibody host were incubated with each blot. Unbound antibodies were washed off in 0.05% Tween-20 in Tris-buffered saline. The LumiGLO peroxidase chemiluminescent substrate kit (SeraCare) was used to detect antibody-labeled proteins, and membranes were imaged using the Bio-Rad ChemiDoc imaging system.