Polyvinylidene fluoride (pvdf)

Western blot was performed as described previously [24 (link)]. In brief, cellular proteins were separated by electrophoresis under appropriate conditions, then electroporated and transferred to polyvinylidene fluoride (PVDF, Millipore, USA) membranes, which were subsequently blocked to prevent non-specific binding. Next, the membranes were incubated with primary antibodies overnight at 4 °C, including SERPINE1 (1:1000, A6211, ABclonal, China), FTO (1:1000, A3861, ABclonal, China), β-actin (1:50,000, AC026, ABclonal, China) and GAPDH (1:2000, AP0063, Bioworlde, USA). After incubation with secondary antibodies for 1 h at room temperature in a darkened environment, we used the Odyssey CLx Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA) to capture images and quantify the band intensities.

Total proteins of plants were extracted with lysis buffer (100 mM Tris–HCl, pH 8.8, 60% SDS, 2% β‐mercaptoethanol). Proteins were separated in a 12% SDS‐PAGE gel, transferred onto polyvinylidene fluoride (Millipore) by wet electroblotting, and detected with primary antibody and anti‐mouse or ‐rabbit (TransGen) secondary antibody. The antigen–antibody complexes were visualised using Immoblion Western Chemiluminescent HRP Substrate (Millipore) under standard conditions. The large subunit of RuBisCO protein served as the loading control. Quantitative analysis of the western blot digital images was performed using ImageJ software.

BCA assay was conducted to measure the concentration of protein, and total protein (30 µg) was separated on SDS-PAGE gel (10%). The proteins were transferred to the membrane of PVDF (Millipore, USA) using a microtransblot device (Bio-Rad, USA). The membrane after BSA solution (5%) blocking was incubated with the primary antibody overnight at 4 °C. It was then treated with an enzyme-labeled secondary antibody (4 °C). GAPDH and β-actin served as internal controls.
Primary reactants: GPX1, GPX2, TrxR1, PI3K, P-AKT, mTOR, eIF4EBP1, Ras, Raf, MEK, ERK1/2, WIPI2, ACOX1, S100A9, iNOS, IL-6, IL-10, NCAM1, APIP, β-actin, and GAPDH (Abcam, USA).
Secondary antibody: Anti-mouse IgG (Cell Signaling Technology, USA).

Total protein in GC tissues and cells were extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, China) adding with protease and phosphatase inhibitor cocktail (Beyotime, China) as described in a previous study(Lin et al. 2019 (link)), separated using Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto polyvinylidene fluoride (Millipore, USA) membranes. Then, the membranes were incubated with primary antibodies (p-S6K, S6K (Abcam, USA), RNFT2 (MyBioSource, USA), Actin, p-S6 and S6 (CST, USA) and secondary antibodies, respectively. The enhanced chemiluminescence (ECL) detection kit (Beyotime, China) was employed to detect chemical signals.

MG63 cells were seeded in a six-well culture plate at a density of 5 × 10⁴ cells/well for 24 h. The cells were treated with β-glycerophosphate (10 mM), l-ascorbic acid (50 µg/mL), or CES (200 µg/mL) in the absence or presence of LDN-193,189 (Smad inhibitor, 100 nM) for 5 days. For signaling pathway analysis, cells were treated with reagents or CES for the indicated time. Whole cell lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors (Sigma-Aldrich). Nuclear extracts were obtained using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, IL, USA), according to the manufacturer’s protocol. The proteins were electrophoresed in a 10% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF, Millipore, MA, USA). After blocking with 5% skim milk for 1 h, membrane was incubated with antibodies against phosphorylated Smad-1 (Ser206, Cat#9553s, Cell signaling technology Inc., MA, USA), Smad-1 (Cat#9743s, Cell signaling technology Inc., MA, USA), phosphorylated Smad-1/5(Ser463/Ser465)/9(Ser465/Ser467) (Cat#13820, Cell signaling technology Inc., MA, USA) phosphorylated p38 (Thr180/Tyr182, Cat#9215s, Cell signaling technology Inc., MA, USA), p38 (Cat#9212s, Cell signaling technology Inc., MA, USA), osterix (Cat#ab209484, Abcam, Cambridge, UK), Smad-1/5/9 (Cat#ab13723, Abcam, Cambridge, UK), laminB1 (Cat#ab16048, Abcam, Cambridge, UK), and β-actin (A5441, 1:20,000; Sigma Aldrich, MO, USA) at 4℃ for overnight. The protein bands were visualized by incubation at room temperature for 1 h with horseradish peroxidase-labeled immunoglobulin G (IgG) antibodies (1:3000; Cell Signaling Technology Inc., MA, USA), and the ECL Western Blotting Detection System (Amersham Biosciences, Little Chalfont, UK).