Western Blot Protein Detection Protocol

Keratinocytes were lysed in 6.5 M urea buffer (85 (link)) supplemented with a mixture of phosphatase (Cocktail 2 and 3, Merck) and protease inhibitors (Merck). Proteins (5–15 μg) were run under reducing conditions using 4–12% precast polyacrylamide gels (Thermo Fisher Scientific, Waltham, MA, USA) or 10% polyacrylamide handmade gels, depending on the probed proteins. Nitrocellulose membranes were incubated with antibodies listed in Supplementary Material, Table S2. Detection was performed using Amersham ECL Prime (GE Healthcare Life Sciences, Little Chalfont, UK). Images were acquired with ChemiDoc™ XRS+ System (Bio-Rad, Hercules, CA, USA). Band density was evaluated using Image Lab Software (Bio-Rad). Glyceraldheyde-3-phosphate dehydrogenase (GAPDH) and vinculin (VCL) were used as loading controls.