Ripa lysis kit

After being treated with 0.2 μM SeNPs@Am (in an equivalent anisomycin concentration level), 0.2 μM anisomycin, or 2 μM SeNPs, the HepG2 cells were washed and lysed by a RIPA Lysis Kit (Beyotime Institute of Biotechnology, China) containing phenylmethylsulfonyl fluoride (PMSF). The protein concentration of cytosolic extract was measured with a BCA Protein Assay Kit (Beyotime). An equal amount of the protein was separated by SDS-PAGE and then transferred onto nitrocellulose membranes (Amersham Biosciences, Pittsburgh, PA, USA). The membranes were blocked with 5 % non-fat milk at room temperature for 1 h and then washed three times with tris buffered saline (TBS) containing 0.05 % Tween 20 for 5 min each time. Thereafter, the membranes were probed at 4 °C overnight with primary antibodies, respectively, that included anti-ICBP90, anti-p16, anti-p21, anti-P-p21(Thr145), anti-p27, anti-P-p27 (Ser10), anti-P-CDK2 (Thr160), anti-p53, anti-P-p53(Ser20), anti-E2F1, anti-p73, anti-P-p73 (Tyr99), anti-Rb, anti-P-Rb (Ser807), and anti-β-actin. Then, the membranes continued to be incubated with relative second antibodies at room temperature for 1 h. The bands were visualized by enhanced chemiluminescence (Cell Signaling Technology, Inc. USA) according to the manufacturer’s instruction. The band density was checked by a FluorChem 8000 system (Alpha Innotech, Santa Clara, CA, USA).

After harvest, cells were treated by RIPA lysis kit combined with the protease inhibitor (Beyotime). Protein concentration of cell lysate was examined by BCA protein assay kit (Takara, Kyoto, Japan). The cell extracts were subsequently separated by SDS-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes (Millipore, MA, United States). After blocking in 5% nonfat milk at room temperature for 2 h, membranes were incubated with primary antibodies overnight at 4°C, following with the treatment by horseradish peroxidase (HRP) coupled secondary antibody (#7076, Cell Signaling Technology) for 1 h at room temperature. Finally, membranes were treated by ECL reagent (Bio-Rad, CA, United States), signals were detected by FluorChem FC3 system (ProteinSimple, CA, United States) and then quantitatively analyzed by ImageJ software as we described before (Xu et al., 2019b (link)).

HCASMCs were cultured in 6-well plates at a density of 5 × 10 ⁵/well. Then, the RIPA Lysis Kit (Beyotime, China) mixed with phosphatase inhibitor (Beyotime, China), protease inhibitor (Beyotime, China), and phenylmethanesulfonyl fluoride (Beyotime, China) at 50× was applied to lyse the HCASMCs. The same amount of total protein was separated on SDS–PAGE gel by electrophoresis and then transferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). Membranes were blocked with blocking buffer (Beyotime, Shanghai, China) at room temperature, and then incubated overnight at 4 ℃ with primary antibodies toMETTL3 (1:1000, Proteintech, Wuhan, China). On the next day, the membranes were incubated in secondary antibodies for 1 h at room temperature. Proteins were imaged using the Tanon-5200 automatic gel imaging system (Tanon, Shanghai, China).

HCASMC cells were distributed in six-well plates at 5 × 10⁵/dish, and the corresponding treatment was carried out for 24 h. Then, cell lysis was conducted with RIPA lysis kit (Beyotime) including phenylmethanesulfonyl fluoride. Quantification of total protein was detected with BCA protein assay kit (Beyotime). Total proteins (20 μg) were separated using 7.5–10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The resolved protein was subsequently transferred onto a nitrocellulose membrane. Membranes were blocked with quick blocking buffer (Beyotime) for 10 min at room temperature, and then incubated with primary antibodies at 4°C for 15 h, including p-STAT3, p-MAPK14, p-AKT (1:1,000; Cell Signaling Technology, Danvers, MA, USA), p-PI3K, PI3K, AKT, mTOR, and p-mTOR (1:1,000; Proteintech Group, Wuhan, China). Membranes were washed and then incubated in secondary antibodies (1:10,000) at room temperature for 1 h. Proteins were imaged by the Bio-Rad Chemi Doc XRS imaging system (Bio-Rad, USA).

THP-1 cells were seeded in 6-well plates at 1.5 × 10⁶ cells/mL. The cells were pretreated with or without LPS (1 μg/mL) for 1 h and then treated with ES (0, 12.5, 25, and 50 μg/mL) for 1 h. After incubation, the cells were harvested by centrifugation (1500 rpm, 5 min). Proteins were extracted by RIPA lysis Kit (Beyotime) containing phenylmethanesulfonyl fluoride (PMSF). Protein concentration in the supernatant was measured using BCA protein assay kit (Takara, Japan). Equal amounts of proteins were loaded on 10% SDS-polyacrylamide gel and transferred onto nitrocellulose membranes (Amersham Biosciences, UK). After blocking in Tris-buffered saline with 0.05% Tween 20 (TBST) containing 5% nonfat milk for 1.5 h, the membranes were incubated with primary antibodies (Cell Signaling Technology) at 4°C overnight. After washing, the membranes were incubated with HRP-conjugated IgG antibodies (Cell Signaling Technology) for 1 h. Membranes were developed using ECL Western blotting detection reagent (Bio-Rad). The band intensity was measured using the FluorChem 8000 system.