P1045

Mouse lung tissues or cultured A549 cells were lysed with RIPA buffer (89900, Thermo Fisher Scientific, USA) containing 1 × protease inhibitor and phosphatase inhibitor (P1045, Beyotime, China). Protein concentrations were measured using the bicinchoninic acid assay (PH0326, Phygene). Equal amounts of proteins (25 or 30 μg) were subjected to gel electrophoresis and transferred onto polyvinylidene fluoridemembranes. These membranes were then blotted with FGF2 (1:1000 dilution, sc-74412, Santa Cruz) or GAPDH (1:1000 dilution, 5174, Cell Signaling Technology, Danvers, Massachusetts, USA) for mouse samples and FGF2 (1:1000 dilution, 20102, Cell Signaling Technology), phospho-p38MAPK (Thr180/Tyr182; 4511, Cell Signaling Technology), phospho-ERK1/2 (Thr202/Tyr204; 4370, Cell Signaling Technology), p38MAPK (8690, Cell Signaling Technology), ERK1/2 (4695, Cell Signaling Technology), phospho-NF-κB p65 (Ser536; 3033, Cell Signaling Technology), phospho-IκBα (Ser32; 2859, Cell Signaling Technology), NF-κB p65 (8242, Cell Signaling Technology), or GAPDH (5174, Cell Signaling Technology) for A549 cell lysates. Subsequently, HRP-conjugated anti-rabbit (1:2000 dilution) or anti-mouse (1:3000 dilution) antibodies were used as secondary antibodies, and enhanced chemiluminescence (1705060, Bio-Rad) was conducted. Densitometry was performed using ImageJ (NIH, Bethesda, Maryland, USA) software.

HUVECs (1 × 10⁵) and HRMECs (1 × 10⁵) seeded into a 6‐well plate were starved for 6 h in 0.5% FBS EBM‐2 and treated with the mixture of VEGF₁₆₅ (50 ng mL⁻¹) and LYTAC gel (2 µM) for 24 and 48 h. The cells were then lysed with RIPA buffer supplemented with protease and phosphatase inhibitor (P1045, Beyotime) to acquire the cellular protein, which was quantified by using the BCA kit (P0010, Beyotime). The samples were mixed with loading buffer, denatured at 100 °C for 5 min, and separated on 4–12% SDS‐PAGE gels (ET12412, ACE Biotechnology). Proteins were then transferred onto polyvinylidene difluoride membranes and blocked with 2% skimmed milk in TBST solution for 1 h at room temperature. After being washed with TBST thrice, the membranes were incubated with primary VEGFR‐2 (1:1000, 2479, CST) or GAPDH (1:20 000, 60004‐1‐Ig, Proteintech) antibodies individually at 4 °C for 24 h. The membranes were then washed with TBST thrice and incubated with secondary antibodies (1:5000, Proteintech) at room temperature for 2 h. Then membranes were further washed with TBST thrice and visualized by ECL detection.

Cells and mitochondria were washed with cold PBS and lysed with RIPA buffer (V900854-100ML, Sigma, USA) containing protease inhibitor cocktails (P1045, Beyotime, China) for 30 min at 4°C. Lysates were centrifuged at 14000 x g for 15 min at 4°C. Lysates were diluted at a ratio of 1 : 4 with protein loading buffer (5 x) (P0015L, Beyotime, China) and denatured at 95°C for 10 min. The protein extracts were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore, USA). The membrane was blocked with Protein Free Rapid Blocking Buffer (PS108P, Epizyme, China) for 30 min at 22°C and incubated overnight at 4°C with primary antibodies. The primary antibodies for the blots were as follows: Tom 20 (1 : 1000, 42406S, CST), complex IV (COX-IV, 1 : 1000, 4844S, CST), TGF-β (1 : 1000, 3711S, CST), and cleaved caspase 3 (1 : 1000, 9664S, CST). Then, the membranes were incubated with secondary antibodies coupled with horseradish peroxidase (1 : 3000, 7074, CST) for 1 h at room temperature. As internal controls, antibody GAPDH (1 : 1000, 5174, CST) was used. For isolated mitochondria, we normalize the bands by controlling the consistent number of platelets in each sample. The chemiluminescence was detected with enhanced chemiluminescence reagent (ECL, WBKLS0500, Merck Millipore, USA) from three times experiments.

HepG-2 cells were pretreated with 200 µg/mL of the WB-H-S1 for 24 h incubation. Total protein was obtained using a lysis buffer (P0013, Beyotime Institute of Biotechnology) with protease and phosphatase inhibitors (P1045, Beyotime Institute of Biotechnology). Next, HepG-2 cells were centrifuged to remove precipitation. Polyacrylamide gels (12%) were used to load the proteins sample, protein separated depend on molecular weight by using SDS-PAGE and the gels were electro-transferred onto polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA). The membranes were blocked by 5% skim milk in TBST and then incubated with the primary antibodies at 4 ℃ overnight. Subsequently, the membranes were washed with TBST three times and treated with the appropriate secondary antibodies for 1 h. Image J software (NIH, Bethesda, MD, USA) was used to analyze the band intensities.

Total protein samples were collected from heart tissues or CFs. Lysate preparation: (RIPA: PMSF: protease inhibitor: phosphatase inhibitor = 100:1:2:2). The following reagents were used: RIPA (P0013C, Beyotime), PMSF (ST506, Beyotime), Protease and phosphatase inhibitor (P1045, Beyotime). The following primary antibodies were used: anti-α-SMA (Proteintech, 14,395-1-AP), anti-TGF-β1 (Proteintech,21,898-1-AP), anti-TGFBR1 (Abcam, ab235178), anti-collagen 1 (Col-1) (Proteintech,14,695-1-AP), anti-p-Smad3 (Cell Signaling Technology, #9520), anti-Smad3 (Cell Signaling Technology, #9523), and anti-GAPDH (Proteintech,60,004-1-lg). Goat anti-rabbit antibody (Ant Gene, ANT020) and Goat anti-mouse antibody (Ant Gene, ANT019) were applied as secondary antibodies.The following primary antibodies were used: anti-α-SMA (Proteintech, 14,395-1-AP), anti-TGF-β1 (Proteintech,21,898-1-AP), anti-TGFBR1 (Abcam, ab235178), anti-Col-1 (Proteintech,14,695-1-AP), anti-p-Smad3 (Cell Signaling Technology, #9520), anti-Smad3 (Cell Signaling Technology, #9523), and anti-GAPDH (Proteintech,60,004-1-lg). Goat anti-rabbit antibody (Ant Gene, ANT020) and Goat anti-mouse antibody (Ant Gene, ANT019) were applied as secondary antibodies.

A549 cells and lung tissue samples were collected, homogenized, and lysed using RIPA lysis buffer (P0013B, Beyotime Biotechnology, Shanghai, China) containing protease and phosphatase inhibitors (P1045, Beyotime Biotechnology, Shanghai, China) for 30 min on ice. After centrifugation at 12000 × g for 15 min at 4 °C, the supernatant was collected, and the protein concentrations were detected using the BCA method (P0010, Beyotime Biotechnology, Shanghai, China). Proteins were separated on SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA), which were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4 °C with primary antibodies against PPARγ (2435, Cell Signaling Technology), PGC-1α (ab191838, Abcam), NRF-1 (ab175932, Abcam), TFAM (ab47517, Abcam), MFN2 (ab124773, Abcam), GRP78 (ab21685, Abcam), CHOP (ab11419, Abcam), Caspase12 (ab8118, Abcam), Caspase3 (9662, Cell Signaling Technology), APN (21613-1-AP, Proteintech), and β-actin (20536-1-AP, Proteintech). After washing thrice, the membranes were incubated with secondary antibodies (SA00001-2, Proteintech) for 1 h at room temperature. The relative intensities of the protein bands were analyzed using the Bio-Rad Quantity One software. The results were normalized to β-actin levels.

Cell lysis buffer for Western and IP (P0013; Beyotime), Protease and phosphatase inhibitor cocktail for general use, 50X (P1045; Beyotime). Cleared lysates were incubated with anti-human IgG magnetic beads to eliminate proteins with nonspecific binding to immunoglobins and beads. The lysates were incubated overnight with Anti-DYKDDDDK-Tag Human mAb (Agarose Conjugated) (M2008; Abmart) at 4 °C. The beads were washed with lysis buffer five times before SDS sample buffer was added to prepare samples for immunoblotting. For mass spectrometry assay, proteins binding to anti-Flag beads were eluted using Flag peptides (F4799; Sigma) and precipitated by trichloroacetic acid. The sample was submitted to the AIMS Scientific Co., Ltd. (Shanghai, China) for mass spectrometry analysis.