Western Blot Protocol for Protein Analysis

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.