Abnormal ATM–p53 axis function in Mule-deficient MEFs. (A) Mule^fl/y MEFs were treated with 1 µM Dox for the indicated times and extracts were immunoblotted to detect Mule. (B) Mule^fl/y and Mule^del/y MEFs were left untreated (−) or treated with 1 µM Dox for the indicated times. Total p53 and phospho-p53-Ser18 (S18-p53) were detected by immunoblotting. (C) Mule^fl/y and Mule^del/y MEFs were treated as in B, and total ATM and phospho-ATM-Ser1987 were detected by immunoblotting. For phospho-ATM, film exposures of 30 s (row 2) and 5 min (row 3) are shown. Vinculin, loading control. (D) Mule^fl/y and Mule^del/y MEFs were treated as in C and total Brca1 was detected by immunoblotting. Film exposures of 2 s (row 1) and 30 s (row 2) are shown. (E) Mule^fl/y and Mule^del/y MEFs were left untreated (−) or treated for 2 h with 1 µM Dox. Lysates were immunoprecipitated with anti-Mule Ab followed by immunoblotting (WB) with anti–phospho-ATM-Ser1987 Ab. Input, total cell lysate subjected to immunoprecipitation with anti-Mule Ab. For A–E, data are representative of two independent experiments.