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. 2008 Apr 14;105(16):6004–6009. doi: 10.1073/pnas.0710748105

Fig. 1.

Fig. 1.

EWS and EWS-FLI1 affect the expression of cyclin D1 isoforms. (A–C) MCF-7 and A673 cells transfected with siGL2 (negative control) and siEWS (A), as well as A673-shEF1 and A673-Ctrl cells grown for 2 days with or without Dox (B), were analyzed for cyclin D1a and D1b mRNA levels by RT-qPCR. The effects of siEWS and shEF1 on the D1b/D1a transcript ratio are plotted in C. (D and E) A673-shEF1 and A673-Ctrl cells grown for 2 days with or without Dox were analyzed by Western blot for cyclin D1a and D1b proteins using the sc-718 and α-D1b antibodies, respectively (D), and by 3′RACE on nuclear RNA using sense primers in intron 4 and exon 5 (E). Nucleotidic positions of polyA sites (pA) in intron 4 and exon 5 are indicated. In addition to the previously reported polyA site at position 571 in intron 4 (15), we identified a novel polyA site at position 1097 (Fig. S4). The detection of transcripts using intron 4 polyA sites required more PCR cycles than that of transcripts using the exon 5 polyA site, in agreement with the low D1b/D1a mRNA ratio.