Supplementary Figure 5: Analysis of splice-site variant in case 102.

One-step RT-PCR confirms that the exon-intron band is preferentially expressed over the exon-exon band in tumor tissue (Panel A). One-step RT-PCR with primers targeting regions upstream and downstream from the mutation site show equal expression, demonstrating continuation of transcription downstream from the mutation (Panel B). Immunoblots are shown in Figure 2b. The exon-exon primers detected weaker bands, reflecting loss of expression in tumor tissues compared with normal tissues in cases with splice-site mutations. The exon-intron primers demonstrated equivalent to greater expression of the retained intron in the tumor tissues. As SMARCA4 introns may be retained in non-cancer tissues, some intronic expression is expected in normal tissues. These data taken together indicate preferential intronic expression, as expected, in cDNA sequenced from tumor samples with splice-site mutations.