Anti scd1

The following antibodies and working concentrations were used: anti-Actin C11 (1:5000, Sigma Aldrich A2066, for western blot), anti-SREBP1 2A4 (1:500, Santa Cruz Biotechnology sc13551, for western blot), anti-SREBP1 H160 (1:100, Santa Cruz Biotechnology sc8984, for immunofluorescence), anti-SREBP2 (1:500, BD Bioscience 557037, for western blot), anti-SCD1 (1:1000, Abcam ab19862, for western blot), anti-GAPDH (6C5) (1:5000, Santa Cruz Biotechnology sc32233, for western blot), anti-AMPK (1:1000, Cell Signalling 2532S, for western blot), anti-AMPK phospho Thr172 (1:1000, Cell Signalling 2531S, for western blot), anti-ACC1 (1:1000, Cell Signalling 3676S, for western blot), anti-ACC1 phospho Ser79 (1:1000, (Cell Signalling 11818S, for western blot), anti-Farnesyl (1:1000, AB4073 Merck Millipore), anti-Hsp90 (1:2000, Santa Cruz Biotechnology sc13119, for western blot), anti-MLC2 (1:1000, Cell Signalling 3675S, for western blot), anti-MLC2 phospho Ser19 (1:500, Cell Signalling 3671S, for western blot and immunofluorescence), anti-FAK C-20 (1:1000, Santa Cruz Biotechnology sc-558, for western blot) and anti-FAK phospho Y397 (1:1000, Abcam ab81298, for western blot).

The cells were lysed in RIPA buffer containing protease and phosphatase inhibitors. The BCA Protein Assay Kit (Pierce, Waltham, MA, USA) was used to determine protein concentrations. SDS-PAGE was used to separate equal quantities of protein, which was then transferred to PVDF membranes (Millipore, Burlington, MA, USA). Membranes were blocked with 5% nonfat milk and incubated with primary antibodies overnight at 4 °C, anti-APOE4 (Abcam, Cambridge, UK; catalog #ab279714,1:1000), anti-ABCA1 (Abcam, Cambridge, UK; catalog #ab18180,1:1000), anti-FAS (Abcam, Cambridge, UK; catalog #ab133619,1:1000), anti-ACC (Abcam, Cambridge, UK; catalog #ab109368,1:1000), anti-SCD1 (Abcam, Cambridge, UK; catalog #ab236868,1:1000), anti-GPT-1 (Abcam, Cambridge, UK; catalog #ab214179,1:1000), anti-SREBP1 (Abcam, Cambridge, UK; catalog #ab313881,1:500), and anti-PPARγ (Abcam, Cambridge, UK; catalog #ab310323,1:1000). Protein bands were detected using an enhanced chemiluminescence (ECL) kit (Thermo Scientific, Waltham, MA, USA) after incubation with suitable horseradish peroxidase-conjugated secondary antibodies.

Total protein was extracted using a RIPA kit (Beyotime Biotechnology, China), separated on polyacrylamide gels, and transferred to PVDF membranes. The membranes were incubated with anti-GK5 (Proteintech Group, IL, USA), anti-EGFR (Cell Signaling Technology (CST), MA, USA), anti-survivin (CST), anti-PLK1 (CST), anti-Bcl2 (CST), anti- cleaved caspase-3 (Asp175, CST), anti-caspase-3 (CST), anti- cleaved PARP (Asp214, CST), anti-PARP (CST), anti-SCD1 (Abcam, MA, USA), anti-SREBP1 (Abcam), and anti-actin (CST) antibodies at 4 °C overnight and were then incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse immunoglobulin G at room temperature for 1 h. Proteins were visualized using Pierce ECL Western blotting substrate and autoradiography. The blots were analyzed using Quantity One 4.6.

Cells were washed in cold PBS and lysed in 50 mM Tris–HCl pH 7.6, 150nM NaCl, 2 mM EDTA and 1 % Nonidet-P40 containing a cocktail of protease inhibitors (Roche). Following a quick vortex, 5× Laemmli sample buffer (300 mM Tris–HCl pH 6.8, 10 % SDS, 50 % glycerol, 25 % β-mercaptoethanol, 0.05 % bromophenol blue) was added and samples were boiled for 10 min. Proteins were quantified by EZQ Protein Quantitation kit (Molecular probe) and cell lysates containing 50 μg of proteins were separated on 4-15 % Criterion TGX precast gels (Bio Rad). The proteins were transferred to PVDF membranes (GE Healthcare) which were then blocked in 10 % non fat dry milk in TBS-Tween. Western blotting was then performed using anti-SCD1 from Abcam (ab19862), and anti-SREBP-1 from BD Technologies (557036) that recognizes the N-terminal domain, including the mature (m) form, of SREBP-1 without distinguishing between the SREBP-1c and SREBP-1a isoforms, and horseradish peroxidase-conjugated secondary antibodies. Horseradish peroxidase-conjugated anti-B-actin was purchased from Sigma-Aldrich (A3854). The immunoblots were visualized using ECL prime (GE Healthcare) and an Alpha Innotech Fluorochem imager (San Leandro, USA).

Cells were collected, washed, and lysed in radioimmunoprecipitation assay buffer (Beyotime, China) containing a protease inhibitor cocktail. Cell lysates (20 μg/lane) were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. Membranes were then kept in 5% skim milk for 1 h and incubated overnight at 4℃ with primary antibodies. Following three washes, the membranes were subjected to incubation with a secondary antibody at room temperature for a duration of 2 h. Visualization was performed using the ECL Plus Ultra-sensitive Chemiluminescence Substrate (Solarbio, China) and a fluorescence and chemiluminescence imaging system (Clinx, China). The primary antibodies used were anti-β-actin (Abcam, UK), anti-GPX4 (Thermo Fisher, USA), anti-SLC7A11 (Thermo Fisher), anti-SCD1 (Abcam), and anti-STK11 (Abcam). The secondary antibody used was goat anti-rabbit IgG H&L (HRP) (Abcam).

The protein levels in maternal placenta and livers were determined by western blot analysis using anti-IL6, anti-TNF-α, anti-IL-1β, anti-PPARα, and anti-ACSL3 (all from ABclonal, Wuhan, China); anti-SCD1 (Abcam, Cambridge, MA); anti-SREBP1c (CST, Danvers, MA); and anti-FASN (CST, Danvers, MA) antibodies. Total proteins were extracted and subsequently separated by 8–12% SDS-PAGE gels. Proteins were transferred onto PVDF membranes and probed with corresponding antibodies overnight at 4°C, followed by incubation with anti-mouse or anti-rabbit secondary antibodies (Beyotime, Shanghai, China) for 1 h at room temperature. The proteins were visualized by using a Western chemiluminescent HRP substrate (Millipore Corporation, United States).