Supporting Information

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Fig. S1. Examination of the mutated transcripts in lymphoblastoid cell lines derived from the patient 1506. (a) Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of the patient with an exon 4 deletion relative to a normal control. A schematic representation of the transcript from exons 3 to 6 of STXBP1 is indicated (top). The exons and primers are depicted as boxes and arrows, respectively. Two PCR products were amplified from the patient's cDNA: the upper was a wild-type (WT) transcript and the lower was the deleted mutant (middle). Only a single WT amplicon was detected in the control. The mutant amplicon was significantly increased by 30 µM cycloheximide (CHX) treatment for 4 h compared to dimethyl sulfoxide treatment as a vehicle control. RT (+): with reverse transcriptase, RT (−): without reverse transcriptase as a negative control. The sequence of the smaller amplicon clearly demonstrated exon 4 deletion (bottom). (b) Quantitative analysis of the nonsense-mediated mRNA decay (NMD) inhibition by CHX based on the data shown in (a). *p = 0.0023 by unpaired two tailed Student's t-test. Averages of duplicated experiments using two distinctive RNA samples are shown with error bars (SD). The mutant transcript lacking exon 4 created a premature stop codon at position 64, and suffered from degradation by NMD in the patient's lymphoblastoid cells. PCR conditions and the primer sequences are available on request.

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CGE_1733_sm_fs1.tif772KSupporting info item

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