Transcripts for Bak isoforms were detected by RT-PCR with primers flanking exon N6 (Physique 1c), thus amplifying both mRNAs with comparable efficiency. NMD substrate, as it is not accumulating by cycloheximide treatment, it has a long lifetime, and even prevention of PTC by interfering with the alternative splicing did not lead to translation of the Bak mRNA. N-Bak protein is also not revealed by proteasome inhibitors. Our data suggest strong translational arrest of N-Bak mRNA in the neurons. We show that this arrest is partially mediated by 5-untranslated region of Bak mRNA and it is not released during mitochondrial apoptosis. translated N-Bak and Bak and on the HeLa cells transiently transfected with the Azaphen dihydrochloride monohydrate respective expression plasmids. Two antibodies (Sigma B-5897 and EMD Millipore 06-536) against the N-terminal region of Bak/N-Bak acknowledged both about 25?kD Bak and about 20?kD N-Bak in either approach (Supplementary Physique 1) and these antibodies were used in the subsequent experiments. Other two antibodies (Ab-2 and G-23) did not show specific binding (data not shown). Antibody Ab-2 has been used8 to demonstrate N-Bak protein in the cortical Azaphen dihydrochloride monohydrate neurons. Why it did not work in our hands is not clear. Immunostaining of the cultured neurons with any of the four anti-Bak antibodies results in high nonspecific background (data not shown). Thus, at least two antibodies specifically detected N-Bak protein when it is expressed from the plasmid. We then compared the levels of transcripts and proteins of Bak isoforms in the mouse brain. The cultured cells used in this study were analyzed as well. Transcripts for Bak isoforms were detected by RT-PCR with primers flanking exon N6 (Physique 1c), thus amplifying both Azaphen dihydrochloride monohydrate mRNAs with comparable efficiency. As shown on Physique 1a, in the newborn (P0) mouse brain the level of N-Bak transcripts (from the neurons) is highly surpassing the level of BH1-3 Bak transcripts (from the non-neuronal cells) that is in agreement with the results of RNase protection assay.6 N-Bak transcripts were easily detected in the Rabbit Polyclonal to NFIL3 cultures of cortical and sympathetic neurons; the low levels of Bak transcripts in these cultures result from the few contaminating non-neuronal cells.6 Rat pheochromocytoma PC6 cells express exceptionally the mRNAs for both Bak isoforms, the levels of N-Bak mRNA being very low. As expected, only Bak but not N-Bak transcripts were detected in the P0 mouse liver tissue and NIH-3T3 mouse fibroblast cells. The proteins were analyzed from the other half of the same samples by immunoblot. In agreement with previous study6 the anti-Bak antibody (EMD Millipore) did not detect N-Bak protein in any of the tissues or cells whereas the Bak protein was always detected (Physique 1b). The same results were obtained with antibody from Sigma, whereas other two antibodies gave again poor results (data not shown). Thus, despite the high levels of N-Bak mRNA the protein was not detected in the neurons, although our assay easily revealed Bak protein from the samples with significantly lower Bak mRNA levels. We conclude that this absence of N-Bak protein in the neurons results from the posttranscriptional regulation rather than minute amounts of the mRNA. We considered three mechanisms that could lead to the absence of N-Bak protein: (i) rapid degradation of the mRNA, (ii) rapid degradation of the protein and (iii) translational block of the mRNA. N-Bak mRNA is not the substrate for NMD Inclusion of exon N to the Bak mRNA generates PTC that corresponds to the 55-nt NMD rule (Physique 1c). Such mRNA could potentially be degraded during the pioneer round of translation thus explaining the absence of N-Bak protein. To test this hypothesis we blocked translation by cycloheximide (CHX) that should lead to rapid accumulation of the NMD substrate mRNAs.10, 11 We treated cultured cortical neurons with CHX for 4 and 8?h Azaphen dihydrochloride monohydrate and determined the levels of N-Bak transcripts by quantitative RT-PCR (qRT-PCR). mRNA for Bax, another Azaphen dihydrochloride monohydrate pro-apoptotic Bcl-2 family member that does not contain PTC was also analyzed.