Halt phosphatase protease inhibitors

Frozen back skin was scraped with a razor blade in order to remove the epidermis, which was ground to a powder as described for RPPA. The powder was then lysed and sonicated for Western blot analysis [16 (link)]. Cultured keratinocytes were lysed in RIPA lysis buffer including 1× HALT phosphatase/protease inhibitors (Invitrogen, Waltham, MA) and briefly sonicated on ice as described [34 (link)]. Protein concentration was determined using the Dc protein assay for both skin and cell culture lysates (BioRad, Hercules, PA). Lysates were separated by SDS PAGE, transferred to PVDF membranes, blocked and probed with primary and secondary antibodies as described [34 (link)]. For keratinocytes, western blots shown are representative of n = 3. For back skins, data are representative of n = 2 (3–4 mice/group). All phospho-protein related images are from fresh blots. Loading controls were performed on relevant stripped blots.

Cultured keratinocytes were lysed in RIPA buffer containing 1× HALT phosphatase/protease inhibitors (Invitrogen, Waltham, MA) and PMSF (Sigma-Aldrich, St. Louis, MO) and briefly sonicated on ice as described (29 (link)). For epidermal lysates, frozen mouse skin was scraped to remove the epidermal layer, which was then ground using a frozen mortar and pestle, placed in skin lysis buffer containing 1× HALT and PMSF and processed as described (26 ). Protein concentrations for epidermal samples were determined using the Dc protein assay (BioRad, Hercules, CA). Samples were separated on SDS-PAGE, transferred to PVDF membranes and processed using established protocols (26 ). Protein bands were visualized using Amersham ECL reagents (GE Healthcare Life Sciences, Pittsburgh, PA). Results are representative of n≥3 for cell culture studies; 3 mice/group for mouse epidermis studies which were repeated at least two times.