Translational efficiency of transcripts generated by ASO-induced skipping of <i>SMN</i> exons in HeLa cells. <i>A,</i>

<p>Diagrammatic representation of <i>SMN</i> pre-mRNA showing annealing positions of ASOs. Exonic and intronic sequences are depicted as in (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049595#pone-0049595-g002" target="_blank">Figure 2A</a>). <b><i>B,</i></b> Splicing pattern of endogenous <i>SMN</i> in HeLa cells treated with various ASOs. Names of ASO used are given on the top of the gel. HeLa cells were transfected with 100 nM of an individual ASO. Cells were collected for total RNA preparation ∼38 hours post transfection. Spliced products were analyzed by RT-PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049595#pone-0049595-g003" target="_blank">Figure 3B</a>. Diagrammatic representation of spliced variants is given on the left of the gel; their sizes are indicated on the right of the gel with names of skipped exons given in brackets. All spliced products represent mixture of transcripts generated from both, <i>SMN1</i> and <i>SMN2</i>. <b><i>C,</i></b> Western blot showing SMN protein isoforms generated from transcripts in panel B. Cells were collected for whole-cell lysate preparation ∼38 hours post transfection. In order to capture the low abundance of SMNΔ3 isoform, we loaded 50 µg of protein from sample treated with E3/I3 Jxn ASO. Protein loading was confirmed by re-probing the blot with anti-β-actin antibodies. Primary antibodies used for probing are indicated on the left of the gel; whereas, SMN isoforms are shown on the right of the gel. SMN is present in all lanes. SMNΔ3 is detectable in lane 2, whereas SMNΔ5 is detectable in lane 3.