Cultured keratinocytes were infected as previously described (Beronja et al., 2010 (link)). Briefly, keratinocytes were plated at 105 cells/well in six-well plates and infected with ∼107 colony-forming units of the appropriate lentiviruses encoding control (shScr;puromycin or shScr;H2B-GFP) or gene-specific shRNA in the presence of 100 µg/ml Polybrene (Sigma-Aldrich).
For Western blot and cell proliferation analyses, cultured keratinocytes were infected with shScr;puromycin (control) or gene-specific shRNA;puromycin. 48 h after infection, cells were selected with 2 µg/ml puromycin (Sigma-Aldrich) for an additional 72 h and then processed (see below). Keratinocytes were cultured in 50 µM Ca2+; 24 h before harvesting, Ca2+ levels were increased to 300 µM. For differentiation assays, cultured keratinocytes were switched into 1.5 mM Ca2+ for 96 h and then analyzed.
For Western blot and cell proliferation analyses, cultured keratinocytes were infected with shScr;puromycin (control) or gene-specific shRNA;puromycin. 48 h after infection, cells were selected with 2 µg/ml puromycin (Sigma-Aldrich) for an additional 72 h and then processed (see below). Keratinocytes were cultured in 50 µM Ca2+; 24 h before harvesting, Ca2+ levels were increased to 300 µM. For differentiation assays, cultured keratinocytes were switched into 1.5 mM Ca2+ for 96 h and then analyzed.
