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The following Supporting information is available for this article:

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.

Additional Supporting information may be found in the online version of this article.

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CGE_1733_sm_fs1.tif772KSupporting info item

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