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. 2010 Sep 14;285(46):35624–35632. doi: 10.1074/jbc.M110.122309

FIGURE 4.

FIGURE 4.

Intron-containing transcripts in cells expressing DHX16-G724N are not subject to NMD and are retained in the nucleus. To inhibit NMD with cycloheximide, HEK293 cells were transfected with wild type DHX16 (Wt) or DHX16-G724N (GN) and then treated with 20 μm of cycloheximide (+CHX) or solvent (−CHX) for 6 h. To inhibit NMD by knocking down UPF1, HEK293T cells were co-transfected for 48 h with a plasmid carrying an shRNA gene (shGFP or shUPF1, at 3 μg for 8 × 105 cells) and a plasmid carrying a DHX16 construct (wild type, mutant G724N, or pEGFP-C1 control (GFP), at 0.3 μg for 8 × 105 cells). A, top, Western blots of protein lysates from G724N-transfected HEK293 cells with or without cycloheximide treatment were probed with anti-DHX16 or anti-GAPDH antibodies. Bottom, RNA from the transfected cells was analyzed by RT-PCR for nPTB exon 10-skipping and exon 10-containing isoforms. B, RT-PCR for the indicated genes was performed with RNA from HEK293 cells transfected with DHX16-G724N and either treated with cycloheximide or not. Intronic/exonic primer pairs were used in all cases, as depicted in the diagram above the gel. C, top three panels, Western blots of protein lysates from HEK293T cells co-transfected with an shRNA-containing plasmid and a DHX16-expressing plasmid were probed with antibodies against UPF1, GAPDH, or DHX16. Bottom three panels, RNA from the co-transfected cells was analyzed by RT-PCR for nPTB exon 10 isoforms, for unspliced DNAJB1 RNA, and for GAPDH mRNA. D, RT-PCR for the indicated genes was performed with RNA from HEK29T cells co-transfected with DHX16-G724N and an shRNA-containing plasmid. Intronic/exonic primer pairs were used in all cases. E, the intron-containing transcripts in cells expressing DHX16-G724N were retained in the nucleus. HEK293 cells transfected with DHX16-G724N and treated with or without cycloheximide were lysed. The lysate was fractionated into two fractions: nuclear (N) and cytoplasmic (C). RNA and protein were isolated from each fraction. Top four panels, RNA was analyzed by RT-PCR for HSPH1, PRPF8, U6, and nPTB RNAs using exonic primer pairs shown as arrowheads; the unspliced and spliced RNAs are indicated (box, exon; line, intron). Bottom panel, Western blots of nuclear and cytoplasmic protein fractions were probed with anti-GAPDH antibody.