Lumiglo peroxidase chemiluminescent substrate kit

Western blots were generated according to a previously described protocol [28 (link)]. Briefly, 5 µL protein lysates were loaded onto SDS-polyacrylamide gels and transferred to Immun-Blot polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA, USA). After blocking in 5% milk, the membranes were incubated first in primary antibodies against p63 (4A4, 1:20,000), COTL1 (Proteintech, 1:10,000), K14 (a gift from Dr. Rose-Anne Romano) [29 (link)], Vimentin (CST, 1:5000), MMP9 (Proteintech, 1:10,000), Fibronectin (SinoBiological, 1:5000), ITGB4 (Proteintech, 1:10,000), E-cadherin (CST, 1:5000), and K6 (a gift from Dr. Julie Segre), then with horseradish peroxidase-conjugated secondary antibodies corresponding to the host of the primary antibody, and then washed in Tris-buffered saline with 0.05% Tween-20. Protein expression was detected with the LumiGLO peroxidase chemiluminescent substrate kit (SeraCare, Milford, MA, USA), and membranes were imaged using a Bio-Rad ChemiDoc imaging system. Uncropped Western blot images can be found in Figures S7–S9.

The transfected cells of one 6-well plate were harvested and lysed in 30 μl Pierce RIPA buffer (Thermo Fisher Scientific) supplemented with 1% benzonase (Novagen, EMD Chemicals) and subsequently incubated for 2 h at RT. Sodium dodecyl sulfate (SDS)-buffer was added to a final concentration of 62.5 mM Tris/HCl, pH 6.8, 100 mM β-mercaptoethanol, 2% SDS, 0.02% bromophenol blue, and 10% glycerol. 30 µl of sample were loaded on a 10% SDS-PA-gel (Laemmli) and run for 1 h at 125 V. In-gel fluorescence was detected using a Fusion FX (Vilber) at λ_480/535 for mVenus detection. Protein samples used solely for immunoblotting were heated for 10 min at 62 °C. Blotted PVDF membranes were blocked with 5% milk in TBS-T and incubated with anti-TAP1 148.3 (hybridoma supernatant generated in-house, 1:10)^{47 (link)} and anti-β-actin (Sigma, clone AC-74, 1:5000). Anti-mouse IgG (Fc specific)-Peroxidase conjugate (Sigma, 1:20,000) was used as secondary antibody. For detection, membranes were incubated with Clarity Western ECL reagent (BioRad) or LumiGLO® Peroxidase Chemiluminescent Substrate Kit (seracare), and chemiluminescence was measured with a Fusion FX (Vilber). Quantification of signal intensities was done with ImageJ (1.52a).

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.