Gradient sodium dodecyl sulfate polyacrylamide gels

Whole cell lysates were prepared from cultured cells by subjecting them to ice-cold lysis buffer supplemented by protease and phosphatase inhibitor cocktails (Sigma). Proteins were isolated and then quantified with Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Total protein samples (30 μg) were subjected to electrophoresis on 7.5%, 10%, and 4% to 15%–gradient sodium dodecyl sulfate polyacrylamide gels (Bio-Rad) and then each was electrophoretically transferred onto a polyvinylidene difluoride membrane (Millipore). Membranes were blocked with non-fat dry milk, washed, and probed overnight at 4 °C with primary antibody followed by horseradish peroxidase (HRP)–conjugated secondary rabbit or mouse antibody (Cell Signaling Technology). All antibodies used in this study are listed in Supplementary Table 1. Bound antibodies were visualized by using an enhanced chemiluminescent HRP antibody detection kit (Denville Scientific).

The cells were lysed using radioimmunoprecipitation assay buffer (RIPA) lysis buffer containing protease inhibitor cocktail (Roche) and phosphatase inhibitors (Roche) on ice for 15 min, the insoluble material was separated by centrifugation (12,000 rpm, 10 min, 4 °C, Centrifuge, Eppendorf, China, 5418R), and the supernatants were collected. The protein concentration was determined by bicinchoninic acid protein (Thermo Fisher Scientific). Equal amounts of protein were subjected to electrophoresis on 10% or 15% gradient sodium dodecyl sulfate-polyacrylamide gels (Bio-Rad) and transferred to nitrocellulose filter membrane (Beyotime). The membranes were blocked with 5% BSA (Beyotime) and incubated with specific antibodies, including p-AKT antibody (CST, USA, 9271, 1:1000), AKT antibody (CST, USA, 9272, 1:1000), GAPDH antibody (CST, USA, 2118, 1:10,000), p-mTOR antibody (CST, USA, 2983, 1:1000), mTOR antibody (CST, USA, 2971, 1:1000), LC3 antibody (proteintech, 14600-1-AP, 1:1000), ACAN (Proteintech, 13880-1-AP, 1:500), MMP13 (Servicebio, GB11247-1, 1:1000), SQSTM1 (Abmart, T59081XS, 1:1000), BECN1 (Abmart, T55092XS, 1:1000), GAPDH (Cell Signaling Technology; 1:10,000), and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Licor 926-32211/926-32210).

Both tissue samples from D-GDM, I-GDM, normal pregnancies and JEG-3 cells were homogenized in RIPA buffer (150 mM NaCl, 50 mM Tris, 1% (v/v) NP-40, pH 7.5). Bradford protein assay and WB analyses were performed as outlined previously.16 17 23 (link) Briefly, 50 µg of protein from placental samples and 30 µg of protein from JEG-3 cells were separated on 7.5%–10% gradient sodium dodecyl sulfate–polyacrylamide gels (Bio-Rad, Mississauga, Ontario, Canada). After electrophoresis, proteins were transferred to methanol-hydrated polyvinylidene difluoride membranes (Immun-Blot PVDF Membrane, Bio-Rad) and blocked with 5% (w/v) non-fat milk dissolved in Tris-buffered saline containing 0.1% (v/v) Tween-20 (TBS-T) for 1 hour at room temperature. After an overnight incubation with primary antibodies at 4°C, membranes were washed in TBS-T and incubated for 1 hour with the secondary antibody. Proteins were visualized by enhanced chemiluminescence reagent (PerkinElmer Inc, Waltham, Massachusetts, USA). Densitometry was conducted using ImageQuant 5.0 software (Molecular Dynamics, GE Healthcare, La Jolla, California, USA) or ImageJ (version 1.50i, National Institutes of Health, USA). Protein levels were corrected for background and normalized to β-actin (ACTB) volumes.