Pcbp1

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.

Antibodies were used against the following: PCBP1 (Cat No. sc-137249, Santa Cruz, WB 1:500); p62/SQSTM 1 (Cat No. 5114, Cell Signaling Technology (CST), WB 1:1000); LC3B (Cat No. 3868, CST, WB 1:1000, IF 1:200); Caspase-8 (Cat No.13423-1-AP, Protein Tech, China, WB 1:500); Caspase-3 (Cat No. 9662, CST, WB 1:1000); PARP (Cat No. 9542, CST, WB 1:1000); c-caspase-3 (Cat No. 9661, CST, WB 1:1000); ULK1 (Cat No.4773, CST, WB 1:1000); c-PARP (Cat No. 9541, CST, WB 1:1000); ATG7 (Cat No.8558, CST, WB 1:2000); ATG12 (Cat No.4180, CST, WB 1:1000); ATG5 (Cat No.2630S, CST, WB 1:1000); β-Actin (Cat No.4967, CST, WB1:2000); anti-mouse HRP-labeled secondary antibody (Cat No. 7076S, CST, WB 1:2000), GAPDH (Cat No. CW0101A, CW Bio-tech, China, WB 1:2000) and anti-mouse HRP-labeled secondary antibody (Cat No. 7076S, CST, WB 1:2000). The western blots protocols were followed as previously reported by our group (Zhang et al., 2016 (link)). Protein band intensity was, respectively, quantified and analyzed with densitometry by using Image J software.