Anti pias4

Cells were fixed with 4% paraformaldehyde for 20 min at room temperature. After the fixation, cells were permeabilized with 0.25% Triton X-100 for 20 min at room temperature and blocked with 3% FBS in PBS for 1 h at room temperature. Cells were then incubated with primary antibodies (1:100, anti-PIAS4, Proteintech, Cat. # 14242-1-AP) diluted in PBS with 3% FBS for 2 h. After washing three times in PBS, the cells were incubated with secondary antibody (1:200, anti-rabbit IgG Alexa fluor 488) for 1 h and followed by DAPI staining. For preimplantation embryos’ IF staining, 0.1% Tween-20 was added in PBS. For PLA, the permeabilized cells were incubated in PBS containing two primary antibodies (1:700, anti-Dppa2, Abcam, Cat. # ab91318; 1:500, anti-Sumo2, Cytoskeleton, Cat. # ASM23) followed by Duolink in situ PLA (Sigma-Aldrich) anti-mouse (minus) and anti-rabbit (plus) probes and detection reagents Green according to manufacturer’s instructions.

Cells were collected and directly lysed in Mammalian Protein Extraction Reagent (Cwbiotech, Cat. # CW0889) with Protease Inhibitor Cocktail (100x, Cwbiotech, Cat. # CW0889). Proteins were quantified following manufacturer’s instructions using Pierce BCA Protein Assay Kit (Thermo Scientific). Equal amounts of proteins were loaded for immunoblotting. Antibodies used were rabbit anti-GAPDH (1:1,000, Bioworld Technology, Cat. # MB001), mouse anti-Flag (1:1,000, Sigma, Cat. # F3165), rabbit anti-HA (1:1,000 CST, Cat. # C29F4), rabbit anti-PIAS4 (1:1,000, anti-PIAS4, Proteintech, Cat. # 14242-1-AP), and rabbit anti-Dppa2 (1:1,000, Abcam, Cat. # ab9138). Anti-rabbit and anti-mouse secondary antibodies were from LI-COR, and membranes were imaged using Odyssey. For anti-HA in Fig 5A and anti-Dppa2 in S4B and S4D Fig, HRP-conjugated anti-rabbit secondary antibodies were used and membranes were imaged using the Western ECL Substrate (Millipore, Cat. # WBKLS0500).