Figure 5. HuR knockdown promotes Fas exon 6 inclusion in 2A^pro-expressing cells.

(A) Workflow: Huh7-T7 cells were transfected with control or HuR siRNA. At 24 hpt the cells were transfected with plasmids expressing GFP or GFP-HuR. Next day the cells were co-transfected with Fas minigene and pTM1-2A, 2AM or empty pTM1 plasmid. Cells were processed for analysis at 3 hpt. (B) Immunoblot analysis from cells processed in (A) using the indicated antibodies (from top to bottom: eIF4GI, HuR, GFP, TIA1, TIAR and GAPDH). (C) RT-PCR analysis of alternatively spliced products from cells processed in (A). (D) Intensity of the bands was calculated by densitometry and the values of ratios between 5–7 and 5–6–7 amplification products were expressed as mean ± s.d. (n = 3; *P<0.05; **P<0.01 by Student’s t-test). The dotted line indicates the ratio of the control sample.