Ripa cell lysis solution

ESCC tissues (n = 20) and their corresponding adjacent tissues (n = 20) were excised during surgery. Sample tissues (100 μg) were homogenized in RIPA cell lysis solution (Solarbio, Beijing, China) and centrifuged at 12 000g for 15 min at 4°C. The supernatants were collected after centrifugation, and their protein concentrations were determined using a BCA protein assay kit (Beyotime Biotechnology， Shanghai，China). The total protein extract was separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane. Western blot analysis was conducted using the anti‐PADI4 antibody (1:2000). All primary and secondary antibodies were diluted in 5% nonfat dry skim milk in TBST (Tris buffered saline, pH 7.6) that contained 0.1% Tween 20. Immunoreactive signals were detected with horseradish peroxidase (HRP)‐conjugated secondary antibodies and visualized using Western blotting luminol reagent (Millipore, Bedford, MA). The blotting images were acquired with an Alpha FluorChem E (ProteinSimple, SantaClara, CA). A second PVDF membrane was prepared using the same protocol as that used with the primary blot, and this second membrane was hybridized with anti‐GAPDH antibody to normalize sample loading.
The expression of ANGPT1, CA9 and TEK in ECA109 cells was measured using a similar protocol.

Cells were first lysed with RIPA cell lysis solution (Solarbio, China) containing protease inhibitor and phosphatase inhibitor (MedChemExpress, USA), the lysate was separated by SDS-PAGE gel and transferred to PVDF membranes (Millipore, USA), then incubated with proteins successively according to the instructions of primary and secondary antibodies. Finally, exposure was performed using the C300 system (Azure Biosystems, USA) in the presence of an ECL luminescent solution (Millipore, USA). For immunoprecipitation, the starting steps were as described above but involved the use of IP lysate (Thermo Fisher Scientific, USA). A portion of the protein solution was taken to directly detect the corresponding protein expression, while the rest of the solution was added to the primary antibody and magnetic beads (Bimake, USA), respectively, according to the manufacturer's instructions. The beads were washed with wash buffer (Thermo Fisher Scientific, USA) and heated to 100°C for 5 min using protein loading buffer (Abclonal, China). Immunoblotting was then performed as described above.