Fig 1: Ectopic expression of KSR1 or KSR2 sustains proliferation of RASless MEFs. (A) Representative images of a colony formation assay in Kraslox cells infected with retroviral vectors expressing the indicated cDNAs in the absence (–4OHT) or presence of 4OHT (+4OHT). (B) Quantification of colony formation in Kraslox and RASless MEFs expressing the indicated cDNAs expressed as percentage of colonies grown in RASless cells compared to those grown in Kraslox cells. Error bars indicate SD (n = 3 independent experiments). (C) Western blot analysis of Flag-tagged KSR1, Flag-tagged KSR2, and KRAS expression in Kraslox MEFs either left untreated (-), treated for 2 weeks with 4OHT (+) to generate RASless MEFs and RASless clones that grew after ectopic expression of KSR1 or KSR2. The expression of KSR proteins was detected using a-Flag, a-KSR1 (long and short exposure) or a-KSR2 antibodies. The presence of 4OHT in the cultures to eliminate the expression of the endogenous KRAS protein is indicated. GAPDH expression served as loading control. The experiment has been repeated at least three times and one representative experiment is shown. (D) Western blot analysis of KRAS, p-MEK1/2, MEK1, pERK1/2 and ERK1/2 expression in Kraslox MEFs, RASless MEFs, or representative RASless clones generated after ectopic expression of shp53-A (shp53), KSR1 or KSR2. The presence of 4OHT in the cultures to eliminate expression of the endogenous KRAS protein is indicated. GAPDH expression served as loading control. The experiment has been repeated at least three times and one representative experiment is shown.