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Fig. 1. Generation of MCF7 stable cell lines in which HEF1 expression is regulated by tetracycline. (A) MCF7 cells were transfected with a plasmid encoding the tTA transactivator and a tetracycline-regulatable expression plasmid either without (CM1,2) or with a HEF1 cDNA insert (HEF1.M1-M5). Total cell lysates (35 µg) derived from uninduced (+ lanes) or induced/mock induced (- lanes) clones were processed by immunoblotting with {alpha}HEF1-SB-R1 antisera to assess HEF1 production levels. The p105 and p115 proteins are differently phosphorylated forms of HEF1 (Law et al., 1998). (B) Lysates (30 µg) were isolated from HEF1.M1 cells induced for the indicated time intervals (in hours, labeled above each lane). Negative controls include lysates isolated after 24 hours from uninduced HEF1.M1 (lane 1) or mock induced CM1 cells (lane 9), probed with antibody {alpha}HEF1-SB-R1, which is specific for HEF1. (C) Lysates from CM.1, HEF1.M1 and HEF1.M2 cells induced for the indicated time intervals (in hours) or maintained in tetracycline (+T) were immunoblotted with antibody to p130Cas (bottom). Corresponding levels of HEF1 at the same time points are also shown (top). (D) Cell lysates from CM1, HEF1.M1 or HEF1.M2 cells prepared at 0, 9 or 24 hours after induction, or in cells maintained in tetracycline (+T) were immunoprecipitated with antibody to p130Cas and then probed with antibody to phosphotyrosine (top), stripped and reprobed with antibody to p130Cas (bottom).