Fig. 4. The kinase activity of c-Abl is important for the stabilization of p53 under basal and stress conditions. (A) Cell extracts were prepared from normal primary fibroblasts (c-Abl positive, lane 1), from c-Abl null fibroblasts (c-Abl negative, lane 2), or from two fibroblastic cell lines that were reconstituted with a c-Abl kinase-defective mutant (K8, lane 3; K10, lane 4). Extracts were subjected to western blotting using anti-p53 antibody (CM5; I) or anti-c-Abl (ABL-148; II). Long exposure of the film was required in order to detect p53 in the c-Abl-negative cells (lane 2), which resulted in an apparent high expression level of p53 in normal cells (lane 1) (I). The amounts of protein loaded were monitored by blotting with anti-α-tubulin (III). (B) Fibroblastic cell lines, either negative for c-Abl (lanes 5–8), or reconstituted with either wild-type c-Abl (c-Abl positive; lanes 1–4) or kinase-defective c-Abl (c-AblK– K8; lanes 9–12), were either untreated (lanes 1, 5, 9) or exposed to γ-radiation (10 Gy) and harvested at the times indicated. The expression levels of p53 were monitored by blotting with anti-p53 antibody (CM5). The amount of protein loaded was determined with anti-histone 2B (II). (C) The three fibroblastic cell lines were treated with mitomycin C (1 µg/ml; lanes 1–3) or doxorubicin (10 µg/ml; lanes 4–6) for 6 h. The expression levels of p53 were monitored as in (A)(I), and the amount of protein loaded was determined with anti-α-tubulin (II). (D) Northern blot analysis of c-Abl null primary fibroblasts (c-Abl negative; lanes 1–4) or normal primary fibroblasts (c-Abl positive; lanes 5–8). Cells were either untreated (NT) or exposed to the proteasome inhibitor ALLN (150 µM for 2 h), mitomycin C (Mito-C; 3 µg/ml for 6 h) or doxorubicin (3 µg/ml for 6 h). Total mRNA was prepared from each sample and subjected to northern blot analysis. The levels of p53 mRNA were determined by probing with 32P-labeled p53 cDNA, and the amount of mRNA loaded was monitored by reprobing with 32P-labeled cDNA for GAPDH.