Fig. 4
Functional assays of the PHF14 missense SNPs. a The log2(θπ ratios), population differentiations (FST), and Tajima’s D of selected regions around gene PHF14 are shown in the top panel. The middle panel shows the structure of the PHF14 protein. A multispecies alignment of the amino acid sequence of PHF14 near the mutation point is provided at the bottom. b Western-blotting analysis of the HEK293 cell lysates transfected with the GFP-tagged recombinant plasmid of WT, I271F, E442D, and I271F, E442D, or the empty vector. WT, I271F, E442D and I271F, and E442D have plasmids overexpressing the wild-type PHF14 protein, the I271F mutant, the E442D mutant protein and I271F, and the E442D double mutant protein, respectively. PEGFP-N1 represents the empty vector. Anti-GFP and anti-tubulin antibodies were used to measure the protein expression of PHF14 and the internal reference protein tubulin, respectively. c The mRNA expression levels of PHF14 in HEK293 overexpressing cell lines measured by qPCR. d Effect of hypoxic stimulus on the apoptosis of HEK293 cells. e HIF-1α and VEGFA expression in the WT, I271F, E442D, and I271F, E442D, or PEGFP-N1 overexpressing HEK293 cells under hypoxic conditions was detected by western blotting. Tubulin was used as the internal loading control. f Gene expression of the cell apoptosis genes, including CASP7, TP53, CDK6, and ATK1 in transfected HEK293 cells, measured by qPCR