Protein extraction and immunoblotting
This protocol is extracted from research article:
Reversible EMT and MET mediate amnion remodeling during pregnancy and labor
Sci Signal, Feb 11, 2020; DOI: 10.1126/scisignal.aay1486

AECs, AMCs, and human and murine tissue were lysed with radioimmunoprecipitation assay lysis buffer [50 mM tris (pH 8.0), 150 mM NaCl, 1% Triton X-100, and 1.0 mM EDTA (pH 8.0), and 0.1% SDS] supplemented with protease and phosphatase inhibitor cocktail and phenylmethylsulfonyl fluoride. After centrifugation at 10,000 rpm for 20 min, the supernatant was collected, and protein concentrations were determined using bicinchoninic acid (Pierce). The protein samples were separated using SDS–polyacrylamide gel electrophoresis on a gradient (4 to 15%) Mini-PROTEAN TGX Precast Gels (Bio-Rad) and transferred to the membrane using iBlot Gel Transfer Device (Thermo Fisher Scientific). Membranes were blocked in 5% nonfat milk in 1× tris-buffered saline–Tween 20 or in 5% bovine serum albumin (BSA) buffer for a minimum of 1 hour at room temperature and then probed (or reprobed) with primary antibody overnight at 4°C. The membrane was incubated with an appropriate secondary antibody conjugated with horseradish peroxidase (HRP) and immunoreactive proteins and then visualized using Luminata Forte Western HRP substrate (Millipore). The stripping protocol followed the instructions of Restore Western Blot Stripping Buffer (Thermo Fisher Scientific). No blots were used more than three times. The following anti-human/mouse antibodies were used for Western blot: N-cadherin (Abcam, ab98952), E-cadherin (Abcam, ab15148), vimentin (Abcam, ab92547), ZEB1 (Novus Biologicals, NBP1-05987), SNAIL (Abcam, ab180714), SLUG (Abcam, ab180714), TWIST (Abcam, ab175430), c-MYC (Cell Signaling Technology, D3N8F), PGRMC1 (Cell Signaling Technology, D6M5M), PGRMC2 (Thermo Fisher Scientific, PA5-59465), and β-actin (Sigma-Aldrich, A5441).

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