Protein Extraction and Analysis from Mouse Skin

The following reagents were purchased from Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A: vinculin, tubulin, p21Waf1/Cip. ASK1 antibodies were purchased from Abcam (Cambridge, MA, U.S.A.) Loricirn and Keratin 1 were purchased from (Biolegend San Diego, CA, USA) and PARP, phospho-p38, p38, Lamin B1, and yH2AX were purchased from Cell Signaling Technology (Beverly, MA, U.S.A). All cell extracts were prepared according to the manufacturer’s instructions for detection of phosphor-ERK (Cell Signaling Technology, Beverly, MA, U.S.A.) as previously described^{21 (link),27 (link)}. Briefly, culture cells were lysed in Laemmli Buffer (Biorad, Hercules, CA, USA) immediately scraped on ice and cell extracts transferred to a microfuge tube; after sonication for 10–15 s to shear DNA, the sample was heated for 5 min at 95 °C and briefly centrifuged. Total extracts were analyzed by SDS-PAGE gel. For whole-skin protein extraction, skin section from 2-day-old newborn mice was frozen in liquid nitrogen and then crushed to powder using a mortar and pestle; pulverized skin was then lysed adding Laemmli Buffer (Biorad, Hercules, CA, USA) and further processed with the cell extract preparation method described above.