Pcbp2

For each ChIP reaction, 2×10⁶ cells were used. Chromatin immunoprecipitation was performed using the Magna ChIP A/G Chromatin Immunoprecipitation kit following manufacturer’s instruction (Sigma) with minor adjustments. Briefly, cells were incubated with dimethyl 3,3′ dithiobispropionimidate-HCl (DTBP) (Thermo Scientific) solution (5 mM) in dark at room temperature for 10 min, then treated with 1% formaldehyde at room temperature for 10 min.^{114 –116 (link)} Then nuclear extracts were collected using NE buffer prepared from the kit. Nuclear extracts were sonicated to generate ~200–500 bp DNA fragments. Nuclear extracts, antibodies, and Protein A/G Magnetic Beads were incubated together at 4°C for overnight, after washes with low salt, high salt, LiCl and TE buffers, bound materials were eluted with the elution buffer. Immunoprecipitated samples were quantified using qPCR. Bound DNA was presented as the percentage of input DNA. Antibodies used for ChIP assays including TDP-43 (Proteintech, 5μg per reaction), Galectin-7 (R&D system, 5μg per reaction) and PCBP2 (Santa Cruz, 5μg per reaction) were listed in key resources table. The primer pair used for ChIP-qPCR of the CLU SNP1 includes forward primer 5′-GGC TGC AGA CTC CCT GAA TC-3′ and reverse primer 5′-GCA AGG GCC CGT TAG AGA AT-3.

HTR-8/SVneo cell lysates from transfected HTR-8/SVneo cells (described above) were prepared using RIPA buffer supplemented with cOmplete protease inhibitor cocktail (Roche) and PhosSTOP phosphatase inhibitor cocktail (Roche). Samples were prepared in LDS sample buffer (Life technologies), run on a NuPAGE 4–12% Bis-Tris Gel (Invitrogen), and subsequently blotted on an Immobilon PVDF membrane. Blotted proteins were visualized using Revert total Protein stain (Li-Cor) according to protocol and measured on the Odyssey Imaging System (Li-Cor). For visualization of specific proteins, the blots were blocked with blocking buffer (Li-Cor) and incubated overnight at 4°C with primary antibodies YBX1 (1:200, Santa Cruz Biotechnology, sc-101198), PCBP1 (1:200, Santa Cruz Biotechnology, sc-137249), or PCBP2 (1:3,000, Santa Cruz Biotechnology, sc-101136). Next, Goat Anti-Mouse secondary antibodies (1:15,000, LI-COR, 926-32210) were used for 1 h at room temperature. Final imaging of the membrane was done on the Odyssey Imaging System.

Embedded first trimester placental explants were sectioned and subjected to standard immunohistochemistry procedures. Antigen retrieval was performed using microwave pre-treatment in sodium citrate buffer. Blocking was done in 5% BSA, followed by overnight primary antibody incubations in 1% BSA at 4 °C. The following primary antibodies were used: YBX1 (1:200, Santa Cruz Biotechnology, sc-101198), PCBP1 (1:200, Santa Cruz Biotechnology, sc-137249), and PCBP2 (1:200, Santa Cruz Biotechnology, sc-101136) that stain for the respective proteins, HLA-G (1:100, Novus Biologicals, NB500-302) that was used as a marker for EVTs, and phosphoH3(Ser10) (1:200, Sigma, 09–797) that was used as a marker for cell proliferation. Finally, Powervision Poly-HRP secondary antibodies were used followed by DAB staining and counterstaining using haematoxilin. ImageJ was used to count the number of proliferating extravillous trophoblasts.