Fig 1: Elevated expression of mitochondrial XPNPEP3 via a reduction of mRNA degradation(A) Western blot analysis of XPNPEP1, XPNPEP2, and XPNPEP3 in lymphoblast cell lines derived from Family A and an age-matched control to patient A. GAPDH was used as a loading control.(B) Quantification of protein expression.(C) Subcellular expression of XPNPEP3 by WB with anti-XPNPEP3, TOM20 (mitochondrial marker), and GAPDH (cytosol marker) antibodies. Total, total cell lysate; Mito, mitochondria; Cyto, cytosol; ctr, control cells; mt, cells with XPNPEP3 variants c.634G>A and c.761G>T.(D) Reverse transcriptase quantitative PCR (RT-qPCR) analysis of XPNPEP3. Total, mRNA level of XPNPEP3; Mito, mRNA levels of mitochondrial XPNPEP3 isoform; Pre, pre-mRNA levels of XPNPEP3.(E) Fitted exponential decay curves of XPNPEP3m mRNA levels in lymphoblast cells. Transcription was blocked by 10 µg/mL actinomycin D.(F) Fitted exponential decay curves of XPNPEP3m mRNA levels in vitro. Oe-WT, wild type of XPNPEP3m transfected back into XPNPEP3-knockout HK-2 cell lines; Oe-MT, XPNPEP3m carrying c.634G>A and c.761G>T transfected back into XPNPEP3-knockout HK-2 cell lines.(G) Protein levels of UPF1, DCP2, G3BP1, and ELAVL1 detected by western blotting. UPF1, regulator of nonsense transcripts 1; DCP2, m7GpppN-mRNA hydrolase; G3BP1, GTPase-activating protein-binding protein 1; ELAVL1, ELAV-like protein 1; KSRP, KH-type splicing regulatory protein.(H) Colocalization of XPNPEP3m RNA with ELAVL1 by combined RNA FISH and immunofluorescence. Bar, 2 µm.(I) Colocalization of XPNPEP3m RNA with KSRP by combined RNA FISH and immunofluorescence. Bar, 2 µm.Data were shown as the mean ± SD of triplicates. Student’s t test was performed between two groups and one-way ANOVA was performed among four groups. *, p < 0.05; **, p < 0.01; ***, p < 0.001. NS, not significant.
Supplier Page from Abcam for Anti-XPNPEP1 antibody