Ethanol-Induced Isoform Shifts in Mcl-1

hNSPs, hNPCs, immature neurons, and mature neurons were exposed to 50 mM EtOH. At 6 and 24 h post-exposure time points, cells were harvested, and total RNA was extracted with an RNA extraction kit (New England Biolabs) according to the manufacturer's instructions. RT-PCR reactions were performed as described previously^{24 (link)}. After treatment with DNase I, followed by phenol/chloroform extraction and EtOH precipitation, cDNAs were synthesized from total RNA using M-MuLV reverse transcriptase. RNA templates were removed by RNase H digestion. A total of 100 ng cDNA was used as template for PCR reactions. Alternatively spliced isoforms of Mcl-1 were amplified by PCR using Mcl-1F: 5′- GGACACAAAGCCAATGGGCAGGT-3′ and Mcl-1R: 5′-GCAAAAGCCAGCAGCACATTCCTGA-3′. β-Actin mRNA was also amplified from the same set of RNA samples by RT-PCR as an internal control using F: 5′-CTACAATGAGCTGCGTGTGGC-3′ and R: 5′-CAGGTCCAGACGCAGGATGGC-3′. Amplified gene products were resolved on a 1.5% DNA-agarose gel.