Ab108424

The liver tissues were fixed with buffered formalin (10%), embedded in paraffin, and cut into 4 µm-thick sections. The slices were incubated with citrate buffer (pH 6.0) for 5 min at 108 °C, pretreated with 3% hydrogen peroxide (H₂O₂) for 15 min at room temperature and washed with PBS. Then, the slices were incubated with normal goat serum for 20 min, followed by incubation with primary antibody against α-SMA (1:500, ab108424, Abcam), Col1α1 (1:50, PA5-36227, Invitrogen) and BECN1 (1:100, ab210498, Abcam) overnight at 4 °C. Besides, the slices were incubated with secondary antibody (1:500, horseradish peroxidase-conjugated anti-rabbit IgG) and the reaction products were visualized with DAB solution.

Total protein was extracted from heart tissue using radioimmunoprecipitation assay lysis buffer (Millipore Corporation, Billerica, MA, USA), and total protein concentration in tissues was determined using bicinchoninic acid kits (Beyotime, Shanghai, China). Protein lysate was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and later transferred onto polyvinylidene fluoride membranes. After 2 h of storage in 5% skim milk, membranes were probed overnight with primary antibodies such as anti-SOX6 (1:500, ab64946; Abcam, Cambridge, MA, USA), anti-collage I (1:1,000, ab138492; Abcam), anti-α-smooth muscle actin (α-SMA; 1:1,000, ab108424; Abcam), anti-transforming growth factor-β1 (TGFβ1; 0.5 μg/mL, ab92486; Abcam), anti-p21 (ab109199, 1/1,000; Abcam), anti-cell division cycle 25 (CDC25; 1:1,000, ab111830; Abcam), and anti-Cyclin B1 (1:200, ab215436; Abcam). Following washing with Tris-buffered saline/Tween 20, the membranes were then incubated with secondary antibody goat anti-rabbit IgG H&L horse radish peroxidase (1:2,000, ab205718; Abcam) for 1 h. Immunoreactive bands were subsequently visualized by enhanced chemiluminescence and later quantified through densitometric analysis (Quantity One; Bio-Rad, Hercules, CA, USA), with GAPDH (1:10,000, ab8245; Abcam) as an internal control.

Ten micrograms of proteins of each group were separated by 11 % SDS-PAGE and transferred onto nitrocellulose membranes. The blots were incubated with primary antibodies against human Nrf2(ab137550, Abcam. 1:400), TRAP-1 (ab2721, Abcam. 1:700), α-SMA (ab108424, Abcam. 1:500), FAP (ab227703, Abcam. 1:600), Collagen I (ab34710, Abcam.1:400), HDAC1 (ab7208, Abcam. 1:500), p-Nrf2 (ab76026, Abcam. 1:300) and β-actin (ab6276, Abcam. 1:2000) overnight at 4 °C, following incubated with the corresponding HRP-conjugated secondary antibody (ab205718, Abcam.1:3500). After washing the membranes twice with Tris Buffered Saline with Tween (TBST, pH = 7.4) and the bands in the membranes were visualized by enhanced chemiluminescence (ECL, Pierce, USA). After incubation for 2 min, the membranes were exposed to x-ray films. The protein bands were scanned and analyzed. The levels of these proteins were calculated as the ratio of the intensity of the specified protein to that of β-actin by using ImageJ2x software (National Institutes of Health, Maryland, USA). The intranuclear Nrf2 protein levels were calculated as the optical density ratio of intranuclear Nrf2 to HDAC1, and the phosphorylated Nrf2 was calculated as optical density ratio of phosphorylated Nrf2 to its total.

Protein samples were collected with the RIPA buffer (Beyotime, China). Commercial kit (Invitrogen, USA) was used for the separation of the proteins in nucleus and cytoplasm. Then, BCA (Beyotime, China) kit was used for the determination of the concentration of proteins. Ten percent SDS PAGE gel was used for the separation of proteins. Then, proteins were transferred to the PVDF membrane (Millipore, USA) and the membranes were blocked with the BSA (Beyotime, China) followed by incubation with the primary antibodies at 4°C overnight. The primary antibodies used in this research were KLF1 (ABCAm, ab175372), E-cadherin (ABCAm, ab40772), Vimentin (ABCAm, ab92547), α-SMA (ABCAm, ab108424), Snail (ABCAm, ab216347), ZBTB7A (ABCAm, ab175918), c-myc (ABCAm, ab32072), β-catenin (ABCAm, ab32572), β-tubulin (ABCAm, ab18207), Histone H3 (ABCAm, ab215728) and β-actin (ABCAm, ab8226) antibodies. These antibodies were diluted with the BSA at the ratio of 1:1000. Then, membranes were incubated with the secondary antibodies for 2 hours. Finally, immunoreactive signals were measured with the western blotting substrate (Millipore, USA). The results of this experiment were analyzed with the Image J.

The LX-2 cells or liver tissues from CCl₄-induced or SSd treatment mice were lysed using RIPA Buffer (Solarbio, Beijing, China), and the total protein concentration was detected by BCA protein assay kit (Cell Signaling Technology, Danvers, MA, USA). The proteins were separated by 10% SDS-PAGE, and then transferred to PVDF membranes (Merck Millipore, Billerica, MA, USA). After blocking with 5% nonfat milk in TBST, the membranes were incubated with primary antibodies against p62 (1:2000, ab109012, Abcam), LC3 (2 µg/ml, ab128025, Abcam), GPER1 (1:1000, ab260033, Abcam), α-SMA (1:1000, ab108424, Abcam) and β-actin (1:5000, ab6276, Abcam) overnight at 4 °C. The blots were incubated with HRP-conjugated secondary anti-rabbit (1:5000) for 1 h at room temperature. Lastly, Immunoreactivities were visualized by chemiluminescence using the ECL kit (Pierce, Rockford, IL, USA). The intensity of protein bands on the Western blot image was quantified using ImageJ software.

The cells or exosomes were lysed with RIPA lysate (Sigma) to obtain proteins. The BCA Assay kit (Solarbio, Beijing, China) was used to determine the protein concentration. An equal amount of protein was added onto 10% SDS-PAGE gels, and the protein was then transferred onto polyvinylidene difluoride membranes (Roche, Basel, Switzerland). The blots were then incubated with primary antibodies, Anti-PI3Kγ antibody (1 : 1,000, ab32089, Abcam), Anti-CD163 antibody (1 : 1,000, ab213612, Abcam), Anti-CD206 antibody (1 : 1,000, sc-58986, Santa Cruz Biotechnology), Anti-PTEN antibody (1 : 1,000, ab267787, Abcam), Anti-AKT antibody (1 : 1,000, ab213612, Abcam), Anti-pAKT antibody (1 : 1,000, ab38449, Abcam), Anti-Vimentin antibody (1 : 1,000, ab20346, Abcam), Anti-α-SMA antibody (1 : 1,000, ab108424, Abcam), Anti-IL-10 antibody (0.5 µg/ml, ab134742, Abcam), Anti-CD63 antibody (1 µg/ml, ab38418, Abcam), Anti-CD61 antibody (1 : 1,000, ab119992, Abcam), Anti-HSP70 antibody (1 : 1,000, ab2787, Abcam), and anti-Actin (1 µg/ml, ab8286, Abcam) overnight at 4°C. The horseradish peroxidase-conjugated secondary antibody was then added to incubate the blots at room temperature for 2 h. At last, the blots were visualized with the Novex™ chemiluminescent substrate reagent kit (Waltham, MA, USA).