Journal: BMC Biology
Article Title: TPST2-mediated tyrosine sulfation orchestrates porcine preimplantation development
doi: 10.1186/s12915-026-02525-7
Figure Lengend Snippet: aTPST2 has an enhancer-like function. A The results of dual-luciferase reporter system. The y-axis shows the construction of luciferase reporter plasmids and LNAs. Three experimental replicates were performed. The error bars represent SEM. One-tailed Student’s t-test was used for the statistical analysis, different letters among a, b, c, and d indicate significant difference ( p < 0.01). B Illustration of aTPST2 deletion mutants. Four different regions are deleted individually, which results in 4 types of aTPST2 mutants: aTPST2 -Δ1, aTPST2 -Δ2, aTPST2 -Δ3, and aTPST2 -Δ4. C Silver staining result following RNA pull-down assay showing the proteins bound to aTPST2 (right) and reverse aTPST2 ( aTPST2 -rev, middle). Only one pull-down assay for mass spectrometry analysis was performed with 3,379 pig 8-cell embryos. One specific band in the right lane was analyzed through mass spectrometry and confirmed as MED8. L, protein ladder. KD, kilodalton. D RT-PCR and western blotting results following RNA pull-down assay using porcine 8-cell embryos. Three experimental replicates were performed. KD, kilodalton. α-, anti-. E Top, aTPST2 and MED8 are stained simultaneously by RNA-FISH combined with immunofluorescence in porcine 8-cell embryos. One nucleus marked by white square is magnified in the right lane. Bottom, line scans of the relative intensity of fluorescence signals indicated by the white dotted line in top panel. Scale bar, 50 μm. F Results of RNA electrophoretic mobility shift assays (REMSA) assays. Three biological replicates were performed. α-, anti-. G Co-immunoprecipitation (IP) followed by RT-PCR and western blotting using porcine embryonic fibroblast (PEF) cells expressing HA-tagged MS2P and MS2-labelled aTPST2 . Three biological replicates were performed. α-, anti-. IP, antibodies used for IP. KD, kilodalton
Article Snippet: For the preparation of the sgRNAs targeting promoters of aTPST2 , the spacer sequences were designed on the website of CRISPR RGEN Tools ( http://www.rgenome.net/cas-designer/ ), and the DNA fragments containing T7 promoter and MS2-labbled sgRNAs were generated from the plasmid sgRNA (MS2) (Addgene, 61,424).
Techniques: Luciferase, One-tailed Test, Silver Staining, Pull Down Assay, Mass Spectrometry, Reverse Transcription Polymerase Chain Reaction, Western Blot, Staining, Immunofluorescence, Fluorescence, Electrophoretic Mobility Shift Assay, Immunoprecipitation, Expressing
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