Enhanced chemiluminescent substrate kit

Cells were collected and lysed with RIPA buffer (Thermo Scientific) supplement with a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Cell lysates was centrifugated at 12,000 g for 15 min at 4 °C, the protein supernatants were collected and quantified using a bicinchoninic acid assay (Thermo Scientific) according to the manufacturers' instruction, then proteins were subjected to SDS-PAGE, and transferred onto PVDF membranes (Millipore, Billerica, USA). after incubated with blocking buffer (5% non-fat dry milk in TBST) for 1 h, PVDF membranes were cultured in indicated primary antibodies overnight at 4 . After three washes with TBST and incubated with HRP-conjugated secondary antibodies for 1 h, antibody detection was conducted using an enhanced chemiluminescent substrate kit (Yeasen).

Modi ed RIPA buffer (50 mM Tris-HCl, pH7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, and 1 mM EDTA) supplemented with protease inhibitors and phosphatase inhibitors (Bimake, Houston, USA) was utilized to lyse cells. Protein concentrations were detected by BCA protein assay reagent (Yeasen, Shanghai, China). Cellular extracts were resolved through SDS-PAGE, transferred to PVDF membranes (Millipore, Billerica, USA), then incubated with the indicated primary antibodies. Enhanced chemiluminescent substrate kit (Yeasen) was used to analyze corresponding antibody speci c signals. Table S5 lists the antibodies used.
Co-immunoprecipitation (Co-IP) 2 × 10 7 RBE and HEK293T cells washed by PBS then were harvested and lysed with NP40 lysis buffer (Solarbio, N8031, Beijing, China) containing protease inhibitors cocktail and phosphatase inhibitors (Bimake, Houston, USA). Then the lysates incubated with Flag-, EGLN3-antibody and control IgG after centrifugation respectively in a rotating incubator overnight at 4 °C. Subsequently, the cell lysates were incubated with Protein A/G (Sigma-Aldrich, St. Louis, MO, USA) for another 3 h. Afterwards, the Protein A/G Dynabeads were eluted and collected. The eluent was boiled and denatured for immunoblotting.

RIPA buffer (Beyotime, China) was used to extract the protein following the appropriate steps. BCA Protein Assay Kit (Beyotime, China) was used to measure the concentration of extracted protein.Cells were collected and lysed with RIPA buffer. Proteins were quanti ed using a bicinchoninic acid assay (Thermo Scienti c), resolved by SDS-PAGE, and transferred onto PVDF membranes (Millipore, Billerica, USA). Antibody detection was conducted using an enhanced chemiluminescent substrate kit (Yeasen).
Antibodies against CEP192 (Bioss, 1:1000, China), E-cadherin (Abcam, 1:500, Cambridgeshire, UK), KRT-8 (Abcam, 1:1000, Cambridgeshire, UK) were used to the related protein level. β-actin (1:1000, Abcam, UK) and GAPDH (1:2500, Abcam, UK) were used for normalization.

For immunoblotting analysis, modi ed RIPA lysis buffer (50 mM Tris-HCl, pH = 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, and 1 mM EDTA) with supplements of protease inhibitors as well as phosphatase inhibitors (Bimake, Houston, USA) were used to lyse cells. The BCA protein assay reagent (Yeasen, Shanghai, China) was used to examinate protein concentrations. Cell extracts were rst decomposed by SDS-PAGE and then applied to PVDF membrane (Billerica millipore, USA). Then, they were incubated by the appropriate primary antibodies. Later, enhanced chemiluminescent substrate kit (Yeasen) was utilized to analyze the speci c signals of indicated antibody. For immunoprecipitaton of endogenous proteins, primary antibodies or control IgG were used to incubate cell extracts in a rotating incubator overnight at temperature of 4°C, then the products were incubated for another 3 h with protein A/G magnetic beads (Sigma-Aldrich, St. Louis, MO, USA). The beads were washed three times using lysis buffer before immunoblotting analysis.