FIGURE 2. Lck–Cre-mediated deletion of Mcl-1 blocks thymocyte development.

a, Number of total thymocytes (mean s.e.m.) from three mice at 8 weeks of age of f/wt or Lck–Cre^f/null. b, Total DN thymocyte subsets (mean s.e.m.) from three mice of the respective genotypes at 8 weeks of age. c, Genomic DNA from sorted DN thymocyte subsets of Lck–Cre^f/wt mice was analysed by PCR to identify the onset of the Mcl-1 allele during development. d, Percentage apoptosis of the DN2 thymocytes was assessed with Annexin-V for the f/wt and Lck–Cre^f/null genotypes. Histograms are representative of three mice analysed at 8 weeks of age. e, MCL-1 protein levels as determined by immunoblots of cultured thymocytes from Rag-2-deficient mice in the absence (Untx) or presence of IL-7 (10 ng ml^-1) for 16 h. f, Mcl-1 mRNA levels in cultured Rag-2-deficient thymocytes after 30, 120 and 360 min of exposure to 10 ng ml^-1 IL-7. The mean s.e.m. is presented of triplicate assays in which Mcl-1 mRNA expression units were normalized to HPRT. g, Isotherms of fluorescein-tagged BIM-BH3 or BAD-BH3 peptides binding to GST–BCL-2 or GST–MCL-1 quantified by fluorescence polarization. Error bars denote s.d. from the mean. h, Mouse thymocyte lysates were immunoprecipitated (IP) with anti-BAD or anti-BIM antibodies, immunoprecipitates were resolved and immunoblots developed with indicated antibodies. i, Mouse thymocyte lysates were incubated with GST–BCL-2 or GST–MCL-1 fusion proteins, complexes resolved and immunoblots developed as indicated.