Figure 3

Several Bim isoforms are expressed in cAMP-stimulated IPC cells. (a) Overview of the Bim gene exons (size in base pairs) with localization of the BH3-domain, start and stop translational codons, sites targeted by the Affymetrix probe sets, and the epitope of the anti-Bim antibody used. (b) Scheme of the Bim mRNA forms detected in IPC cells exposed to 8-CPT-cAMP (100 μM) for 2 h. The known major isoforms (BimEL/L/S), Bim β4, the novel variant η-BimL with an additional exon downstream of the BH3 encoding exon, were sequenced from amplified cDNA. Two additional variants (η-BimEL and η-BimS) were detected by RT-PCR (see Supplementary Table S7a and Materials and Methods for details). The predicted MW (right column) is shown for each variant. (c) The level of the six BH3-containing Bim transcript variants was estimated by qRT-PCR, using specific primer pairs. The fold induction is shown after 3 h incubation with 8-CPT-cAMP (200 μM), when the expression relative to the BimL transcript reached 65% for BimEL and for BimS, and 5, 11, and 0.1% for the novel variants η-BimEL, η-BimL, and η-BimS, respectively. The results are the mean of three independent experiments±S.E.M. (d) The fold change in Bim-transcript after 2 h incubation with N⁶-MB-cAMP, based on hybridization to the three microarray probe sets (color code as in panel a). Note the blunted response to N⁶-MB-cAMP in the IPC_ICER, but not the IPC_BCL2 cells