Fig. 1. Raf activation in primary mouse keratinocytes induces cell cycle arrest and differentiation. (A) Expression of RafER in primary mouse keratinocytes infected with RafER and empty vector (LXSN) retroviruses. Protein (20 µg) was separated on SDS-12% polyacrylamide gels and probed with with antibodies specific for ER or actin. (B) RafER or LXSN infected keratinocytes were lysed at the indicated time points following addition of 200 nM 4-hydroxytamoxifen (OHT). The lysates were normalized for protein content followed by western blotting using either an anti-phospho-MAP kinase or a p42ERK2 antibody. (C) RafER or LXSN infected keratinocytes were analysed for BrdU incorporation in triplicate via LSC at different time points after addition of OHT. (D) RafER infected keratinocytes were cultured in the absence or presence of OHT for 24 h. Cells were analysed for BrdU uptake and examined for DNA content, by propidium iodide staining. (E) Protein lysates were prepared from RafER infected keratinocytes after addition of OHT at the time points indicated. These were analysed by western blotting with antibodies specific for involucrin, keratin 1 and actin. Multiple forms of murine involucrin are detected (Li et al., 2000).