Anti erk1 2

Protein extracts were boiled in RIPA buffer (Beyotime) and separated by sodium dodecyl sulfate–polyacrylamide electrophoresis gel electrophoresis (SDS‐PAGE). The proteins were then transferred to polyvinylidene fluoride membranes (Millipore) and probed with anti‐CD9, anti‐CD63, anti‐ALIX, anti‐TSG101, anti‐EGFR, anti‐FXR1, anti‐TLR‐4, anti‐MMP2, anti‐TGF‐β1, anti‐phospho‐STAT5, anti‐STAT5, anti‐phospho‐ERK1/2, anti‐ERK1/2, anti‐phospho‐AKT, anti‐AKT, anti‐Ki‐67, anti‐PTEN (Abcam), anti‐Bcl‐2, anti‐Bax, anti‐E‐Cadherin, anti‐Vimentin, and anti‐GAPDH antibodies (Cell Signaling Technology). Electrochemiluminescence (Millipore) was applied to determine protein expression levels.

The lung tissues from the experimental rats and lung fibroblasts treated with Chol-HCQ were homogenized in RIPA lysis buffer (Beyotime Biotech, China) containing 1 mM phenylmethylsuphonyl fluoride. The lysates were then centrifuged at 13,000 rpm for 15 min at 4 °C and the supernatants were collected and stored at −80 °C. A BCA protein assay kit (Pierce, Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to determine the protein concentrations. Equal amounts of protein were loaded and run on 10% SDS-PAGE gels, transferred onto Millipore PVDF membranes and blocked with 4% BSA. Then, the membranes were incubated with primary antibodies at 4 °C. The following primary antibodies were used: (1) anti-NF-κB (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), (2) anti-phospho-NF-κB (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), (3) anti-ERK1/2 (AbcamPLC, Boston, MA, USA), and (4) anti-phospho-ERK1/2 (Thr202/tyr204) (AbcamPLC, Boston, MA, USA). Antibodies were detected with horseradish peroxidase (HRP)-conjugated secondary antibody and the blots were developed with the ECL-Plus reagent (Millipore, MA, USA). The blots were tested for GAPDH (AbcamPLC, Boston, MA, USA) to confirm equal protein loading.

Total proteins were isolated from whole larvae at 5 dpf using RIPA buffer supplemented with complete EDTA-free Protease Inhibitor Cocktail (Roche, Mannheim, Germany). Total proteins were separated on SDS-polyacrylamide gels and transferred onto PVDF membranes (Immobilon-P; Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat dried milk in TBST (20 mM Tris-HCl (pH 7.5), 500 mM NaCl, and 0.1% Tween-20) for 1 h at room temperature and then blotted overnight with primary antibodies at 4°C. After washing with TBST, the membranes were blotted with horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc.) for 1 h at room temperature. The reactions were detected using ECL Prime Western Blotting Detection Reagent (GE Healthcare, Wauwatosa, WI, USA). The following antibodies were used as primary antibodies: anti-cleaved caspase-3 (1:500 dilution; Cell Signaling Technology Inc.), anti-H3K27me3 (1:1,000 dilution; Abcam), anti-ERK1/2 (1:1,000 dilution; Abcam), and anti-phosphorylated ERK1/2 (p-ERK1/2) (1:1,000 dilution; Abcam). All figures showing quantitative analysis include data from at least three independent experiments. Quantitative analysis of the western blot data was carried out using Photoshop software (Adobe).