Fig. 5.
Effect of asEPO-R cDNA transfection on EPO-R protein expression in cultured HEK-293 cells. (A) Cells were cotransfected with sEPO-R-FLAG cDNA (2 μg) and variable amounts of asORF2-long cDNA and immunoblotted for EPO-R, FLAG, and β-actin. *, P < 0.05 vs. 0 μg of asEPO-R cDNA. (B) Cells were cotransfected with asORF2-long cDNA (0–2 μg) and variable amounts of sEPO-R-FLAG cDNA and immunoblotted for FLAG and β-actin. *, P < 0.05, 2 μg vs. 0 μg asORF2-long at the same sEPO-R-FLAG cDNA level. For A and B, three independent experiments were performed (mean ± SD, one-way ANOVA). (C) Immunocytochemistry of transfected cells shows increased EPO-R production in response to sEPO-R-FLAG and asORF2-long cotransfection. (Left) Whole-cell images of transfection with vector only, asORF2-long only, sEPO-R-FLAG only, or sEPO-R-FLAG + asORF2-long cDNAs, stained with anti-FLAG antibody for EPO-R-FLAG (red) and phalloidin for β-actin (green). (Scale bars, 20 μm.) (Right) Confocal optical section of cells cotransfected with sEPO-R-FLAG + asORF2-long cDNAs, stained for EPO-R(FLAG) and β-actin. (Scale bar, 10 μm.) (D) Cultured cells were cotransfected with sEPO-R-FLAG cDNA (2 μg) and different antisense cDNAs (2 μg): asORF2-long, antisense cDNA with ≈300 bp + ORF2 coding region; asORF2-short, antisense cDNA spanning the putative ORF2 coding region; asORF2-shortmut, asORF2-short with mutated start codon; asORF1, antisense cDNA spanning the putative ORF1 coding region; asORF1mut, asORF1 with mutated start codon. EPO-R-FLAG expression was detected by immunoblot with anti-FLAG normalized to β-actin. A typical blot (Upper) and average data from four independent experiments (Lower) are shown. The mean EPO-R-FLAG/β-actin expression with vector only was arbitrarily set at 100%. Mean ± SD, one-way ANOVA: P < 0.05 * vs. empty vector † vs. corresponding wild-type antisense cDNA.