Figure 7
RT-PCR analysis of cytoplasmic and nuclear RNAs. Cytoplasmic (C) and nuclear (N) RNAs were isolated by using the PARIS™ Kit (Ambion) according to the manufacturer's protocol. RT-PCR conditions were as in Fig. 1. The RT-PCR product of 120 nt amplified by the E3U-I3L primer pair (see Fig. 1) indicates the presence of the c-fos-2 transcript in both cytoplasmic and nuclear fractions. Amplification of the unspliced fosB transcript (I4U-I4L primers) was included for comparison. Transcript coding for glutathione peroxidase 1 was amplified as control of efficient cellular fractionation. Primer UGPX1 (5'-GCAGAAGCGTCTGGGACCTCGTG) was located in gpx1 exon 1, and primer LGPX1 (5'-GGGAATTCAGAATCTCTTCATTCT TGCCA) in gpx1 exon 2 (the intron between both exons is 218 nt long). UGPX1- LGPX1 primers generated a single RT-PCR product of 101 nt when using the cytoplasmic RNA as template, indicating no detectable contamination with nuclear gpx1 pre-mRNA. In contrast when using the nuclear RNA as template, a fragment 319 nt long was detected in addition to the expected product from spliced gpx1 mRNA. Since the Ambion's PARIS™ Kit is designed for the isolation of both RNA and protein from the same sample, efficient cellular fractionation was further confirmed by Western blot analysis (botton panel) with an Ab specific for the cytoplasmic glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). GAPDH was present only in the cytoplasmic fraction, further indicating no detectable contamination with the nuclear fraction.