Ab179520

Cell lysate was electrophoresed and transferred to a polyvinyllidene difluoride (PVDF) sheet (Sigma-Aldrich, St. Louis, MO, USA). The membranes were nonspecifically blocked in a 1% skim milk solution and incubated with primary antibodies for PADI2 (ab56928), PADI4 (ab128086), and citrullinated histone 3 (cit-H3; ab5103), caspase-2 (ab179520) (Abcam, Cambridge, UK), caspase-3 (9662), cleaved caspase-3 (9661), caspase-8 (9746), cleaved caspase-8 (9496), caspase-9 (9502), cleaved caspase-9 (7237), p21-activated kinase 1 (PAK1) (2602), focal adhesion kinase (FAK) (3285), phospho-FAK(8556), paxillin (12065), phospho-paxillin (2541), p65 (8242), and heat shock protein 90 (hsp90) (4874) (Cell Signaling Technology, Danvers, MA, USA) followed by the respective HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Blotting was visualized by chemiluminescence reaction (ECL; GE Healthcare, Little Chalfont, UK). The respective band intensities were measured using ImageJ (version 1.42; http://rsb.info.nih.gov/ij).

Total proteins were extracted from Swan 71 cells, villous tissues, or mouse placental tissues with RIPA lysis buffer on ice for 30 min. Protein concentrations were determined using BCA Protein Quantification Kit (Vazyme, Nanjing, China). Western blotting analysis was performed as described previously [62 (link)]. Primary antibodies included anti-β-actin (ab8226, 1:5000), anti-β-Tubulin (ab108342, 1:10000), anti-GAPDH (ab8245,1: 5000), anti-Caspase-3 (ab184787,1:1000), anti-Bcl-2 (ab182858, 1:1000), and anti-Caspase-2 (ab179520, 1:2500), all of which were purchased from Abcam (Cambridge, UK). Secondary antibodies included goat anti-rabbit immunoglobulin G (IgG) (dilution 1:10,000; ab207995, Abcam) and goat anti-mouse IgG (dilution 1:10,000; ab207996, Abcam). The intensities of Western blotting bands were quantified using Image J software with β-actin, Tubulin, or GAPDH as loading control. In some cases, proteins with different molecule weights were analyzed in one gel and one membrane. Subsequently, this membrane was cut into different parts based on the locations of protein bands and then stained with their corresponding antibody. Loading controls with different molecular weights from the target proteins were selected to avoid overlap or proximity. For clarity, only one loading control was shown in figures.