Expression of the introduced mcl-1 gene product in FDC-P1 transfectants. FDC-P1 clones transfected with either the inducible pMAMmcl-1 vector (A and C), the constitutively expressing pCMVmcl-1 vector (B and D), or appropriate control vectors (no mcl-1 insert) were assayed for expression of the introduced Mcl-1 gene product (A, B, and D) or expression of bcl-2 (B) by Western blot. (A) mcl-1 transfectant 7.5-MAMmcl-1 (lanes 1 through 4) and vector-only clone 8.5-MAMneo (lanes 5 and 6) were either exposed (+) or not exposed (−) to dexamethasone and assayed at 2 hours. Lysate from TPA-induced ML-1 cells (5 × 10⁻¹⁰ mol/L TPA for 3 hours) was included on the blot for comparative purposes (lane 7). Each lane contains 40 μg of protein. Either preimmune serum (lane 1, Pre.) or anti–mcl-1 immune serum (affinity-purified, lanes 2 through 7) was used. In lane 3, the immune serum had been blocked (Bl.) with bacterially produced mcl-1–GST fusion protein. (B) mcl-1 transfectants S3-CMVmcl-1 and S8-CMVmcl-1 and vector-only clones V1-CMVneo and V2-CMVneo were assayed for expression of Mcl-1 as in (A), except that an IgG fraction of the anti–mcl-1 antiserum was used. The lysate in lanes 1 through 4 represents 2 × 10⁶ cell equivalents and that in lane 5 represents 2 × 10⁵ cell equivalents. The numbers on the left indicate molecular weight markers (in kilodaltons). (C) The transfectant clones shown in (A) were assayed for expression of the endogenous mouse bcl-2 gene produce. Each lane represent 1 × 10⁶ cell equivalents. (D) The S3-CMVmcl-1 transfectant was used for immunoprecipitation of either mcl-1 (lanes 1 and 4; 8 × 10⁶ cell equivalents per lane) or bcl-2 (lane 2). Lane 3 contains lysate (no immunoprecipitation) from TPA-treated ML-1 cells (10⁶ cells as in [A], lane 7), which had previously been found to express abundant levels of Bax. After immunoprecipitation, Western blotting was performed using an anti-bax antibody (lanes 1 through 3) or the anti-bax antibody after preincubation with the peptide against which the antibody had been generated. (E) The transfectant clones shown in (B) were assayed for expression of the introduced Mcl-1 gene product after exposure for 1 day to 20 μg/mL etoposide (lanes 1 through 3) or after incubation for 3 days to medium lacking IL-3 and FBS (lanes 5 through 7). The number of cell equivalents loaded was 4 × 10⁶ in lanes 1 through 3 and 2 × 10⁶ in lanes 5 through 7. ML-1 cells treated with TPA (5 × 10⁻¹⁰ mol/L for 3 hours) were included on the blot as a positive control (2 × 10⁵ cell equivalents; lanes 4 and 8).