Fig 2.
SMN deficiency has no effects on trans-acting regulators of exon 7 splicing. (A) Western blot analysis of wild-type (Control) and NIH 3T3-SmnRNAi cells cultured for 5 days without (−) or with (+) Dox. Blots were probed with antibodies against proteins that have been previously implicated in the regulation of SMN exon 7 splicing. Increasing amounts of protein extract from wild-type NIH 3T3 cells (12.5%, 25%, 50%, and 100%, respectively) were loaded on the left side of the blot as a reference for protein quantification. Gemin8 was used as positive control for a protein whose expression is affected by SMN deficiency (11) and GAPDH as a loading control. (B) Schematic representation of the SMN1 and SMN2 RNA probes used in pulldown assays. The probes encompass the last 68 nucleotides of SMN intron 6, the entire exon 7, and the first 25 nucleotides of intron 7. The nucleotide difference between the SMN1 (C) and SMN2 (T) RNAs as well as the splice junctions (GT and AG) and the branch point adenosine (A) are indicated. (C) Biotinylated RNA probes (SMN1 and SMN2) or no RNA probe (mock) were incubated with whole-cell extracts from NIH 3T3-SmnRNAi cells grown in culture for 5 days without (−) or with (+) Dox. RNA-protein complexes were purified on streptavidin agarose, and bound proteins were analyzed by Western blotting with the indicated antibodies. Input represents 10% of the total amount of protein extract used in the assay.