Western ecl substrate

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).

To denature proteins, cell lysates were added to 5× loading buffer (Beijing TDY Biotech) and heated to 95°C for 5 min. Protein samples were separated by SDS-PAGE electrophoresis, transferred semi-dry onto NC membranes (Millipore), and blocked in Tris-buffered saline-Tween 20 (TBST) containing 5% nonfat milk for 30 min, after which the immunoblotting was performed by incubating with the primary antibody for 10 min at room temperature, and then overnight at 4°C. After being subjected to 5 washes, the membranes were incubated with goat anti-mouse/rabbit IgG (H+L)-HRP secondary antibody (Beijing TDY Biotech, 1:10000 dilution) for 40 min and were subsequently exposed to light using western ECL Substrate (Millipore). The relative expression levels of each protein were assessed using ImageJ software. Primary antibodies used in this study are listed in Supplementary Table 2.