The human β-globin genes expressed in <i>Drosophila</i> S2 cells.

(A) Schematic representation of the β-globin transcripts expressed in S2 cells. The grey boxes represent the coding β-globin sequences. The white boxes indicate the 5′ UTR and 3′ UTR of the pMT vector (see Materials and Methods S1 for details). The box marked V5 indicates the position of the V5 tag. The mut RNA carries a shorter intron 2 and mutations in both the 5′ and 3′ splice sites of intron 2, as indicated in the figure. (B) Western blot analysis of the expression of the wt and mut β-globin genes. The expression of the β-globin genes was induced with 500 µM CuSO4 for 24 h and analyzed by Western blotting using the anti-V5 antibody. Protein expression was detected only from the wt construct. As a loading reference, a section of the PVDF filter containing proteins in the 50–90 kDa range was stained for total protein with Coomassie blue. (C) The expression of the β-globin genes analyzed by immunofluorescence. Expression of the wt and mut β-globin genes was induced as described above and the cells were stained with the anti-V5 antibody (red) to visualize β-globin expression. DAPI counterstaining was used to visualize the nuclei (blue). Exposure times were the same for all images. The magnification bar represents 5 µm. (D) The β-globin transcripts analyzed by RT-PCR. The expression of the wt and mut β-globin genes was induced as described above. Total RNA was purified and reverse-transcribed, and the β-globin sequences were amplified by PCR primers flanking intron 2, as indicated in the figure (lanes 2 and 4). The genomic DNA isolated from β-globin wt or mut S2 cells was used in parallel to check the splicing pattern (gDNA, lanes 1 and 3). Molecular mass standards are shown (M) and the length of the major bands is indicated in bp.