Figure legends
Figure 1. Schematic representation of the minigene splicing assay constructed by pSPL3 vector and position of presumed missense variants we studied in SLC12A3 gene. Panel (A): Transcription begins at the SV40 promoter and ends at the LPAS (late poly(A) signal). The pSPL3 vector contains two exons (SD and SA) and a functional intron. The target exon with partial flanking introns was inserted into pSPL3 vector via XhoI and NheI cloning sites to form the wild-type or mutant plasmid. MCS, multiple cloning sites. Panel (B): Position of 10 variants. Orange boxes and black lines between them respectively indicate the coding exons and introns sequences and their sizes are out of proportion. The BDGP scores of donor and acceptor splice sites are represented in decimal, as shown at the bottom.
Figure 2. Agarose gel electrophoresis of RT‐PCR products expressed from the SLC12A3 minigenes in HEK 293T and Hela cells, respectively. (A, C) Gel electrophoresis with desired results in HEK 293T and Hela cells, respectively. Lane 1: marker; Lane 2: empty vector pSPL3 (263bp); Line 3: pSPL3 Ex5 (403bp (140bp + 263bp) and 263bp); Line 4: c.602G>A (403bp and 263bp); Line 5: c.602G>T (263bp); Line 6: pSPL3 Ex13 (365bp (102bp + 263bp) and 263bp); Line 7: c.1667C>T (365bp and 263bp); Line 8: pSPL3 Ex15 (363bp (100bp + 263bp) and 263bp); Line 9: c.1925G>A (263bp); Line 10: pSPL3 Ex21 (365bp (102bp + 263bp)); Line 11: c.2548G>C (263bp); Line 12: pSPL3 Ex22 (375bp (112bp + 263bp)); Line 13: c.2549G>C (375bp and 263bp). (B, D) Gel electrophoresis with unpredicted results in HEK 293T and Hela cells, respectively. Lane 1: marker; Lane 2: empty vector pSPL3 (263bp); Line 3: pSPL3 Ex24 (387bp (124bp + 263bp)); Line 4: c.2755A>T (387bp); Line 5: c.2863A>T (387bp); Line 6: pSPL3 Ex12 (263bp); Line 7: c.1452C>G (263bp); Line 8: c.1567G>A (263bp).
Figure 3. Statistical analysis of RT‐PCR products in HEK 293T (A) and Hela cells (B).The percentage of exon exclusion (%) = (lower band / [lower band + upper band]) × 100. Error bars represent SEM (n=3). *p < 0.05; ****p < 0.0001, independent-samples T test.