Figure 3.

The 5′ half of the Ig-μ minigene VDJ exon is necessary for efficient NMD of Ig-μ/Sxl hybrid transcripts. (A) Relative hybrid mRNA levels of HeLa cells stably expressing the constructs schematically represented on the left were determined by real-time RT–PCR and are shown in the right panel. For each pair of hybrid constructs, the PTC− mRNA level (white bars) was set as 100% and the PTC+ mRNA level (gray bars) was calculated relative to it. The hybrid mRNA values were normalized to endogenous GAPDH mRNA. Average values of three real-time PCR runs of a typical cell pool are shown, and error bars indicate standard deviations. In the left panel, exons originating from Ig-μ are depicted in black and Sxl exons are depicted in gray. The position of the PTC is indicated by an asterisk and corresponds to ter310 in miniμ or ter43 in Sxl, respectively. The white regions labeled with Δ mark deletions. (B) Northern blot analysis of 15 μg of total cellular RNA isolated from HeLa cells stably transfected with the indicated hybrid constructs. As a loading control, the 18S rRNA band from the ethidium bromide-stained gel before blotting is shown in the lower panel.