Rabbit anti cleaved caspase 3

CHI was kindly provided by Shenzhen Chipscreen Biosciences, Ltd (Shenzhen, China). BTZ was obtained from BSP Pharmaceuticals S.p.A (Lazio, ITA), dissolved in saline solution as a stock solution for 5 mmol/L, aliquoted and stored at -80 °C. The following antibodies were used: rabbit anti-HDAC1 (catalog #AF0178), rabbit anti-cleaved Caspase-3 (catalog #AF7022), rabbit anti-cleaved PARP (catalog #AF7023), rabbit anti-cleaved Caspase-8 (cleaved-Asp384; catalog #AF5267), rabbit anti-γ-H2AX (Tyr143; catalog #AF8482), mouse monoclonal anti-β-actin (catalog #T0022) and goat anti-rabbit Alexa Fluor 594 (catalog #S0006) were purchased from Affinity Biosciences (Cincinnati, OH, USA). Rabbit anti-Ki67 (catalog #GB13030-2) was purchased from Servicebio (Woburn, MA). Matrigel (catalog #356237) was purchased from BD Biosciences Discovery Labware (Two Oak Park, Bedford, MA).

Tissues or cell suspensions were collected for lysis and protein extraction, and the protein concentration was determined by a BCA protein assay kit (Abcam, Shanghai, China). Samples were electrophoresed in 8–12% SDS-PAGE gels, transferred to PVDF membranes (Millipore, Hayward, CA, USA), and then incubated with primary antibodies including rabbit anti-Nrf2 (1:1000, Affinity, Changzhou, China, AF7904), mouse anti-β actin (1:10,000, Affinity, Changzhou, China, T0022), rabbit anti-Bcl-2 (1:1000, Affinity, Changzhou, China, AF6139), rabbit anti-Bax (1:1000, Affinity, Changzhou, China, AF0120), rabbit anti-Cleaved-caspase3 (1:1000, Affinity, Changzhou, China, AF7022), rabbit anti-LC3 (1:1000, Affinity, Changzhou, China, AF5402), rabbit anti-Beclin1 (1:1000, Affinity, Changzhou, China, AF5128), rabbit anti-p62 (1:1000, Affinity, Changzhou, China, AF5384), rabbit anti-NF-κB (1:1000, Affinity, Changzhou, China, AF5006), rabbit anti-PPARγ (1:1000, Affinity, Changzhou, China, AF6284), and rabbit anti-Lamin B (1:10,000, proteintech, Wuhan, China, 12987-1-AP) at 4 °C overnight and HRP-conjugated secondary antibody (proteintech, Wuhan, China, 1:5000) at room temperature for 1 h. Target bands were detected using an enhanced chemiluminescence system, and quantitative analyses were performed using the Image J software (v 1.48, National Institutes of Health, NIH, USA).

The liver tissues and LX2 cells were washed, homogenized on ice with RIPA buffer (Sigma-Aldrich, Saint Louis, MO, USA), and centrifuged at 8000 rpm for 10 min. The protein concentration in the supernatant was determined using a Bradford assay. Lysates containing equal amounts of protein were separated by SDS-PAGE. The western blot analysis was performed as previously described^{21 (link)}. The following primary antibodies were used in this study: rabbit anti-BCL2L10 (1:500, Origene), rabbit anti-glyceraldehyde phosphate dehydrogenase (GAPDH; 1:8000, Cell Signaling Technology), rabbit anti-cleaved caspase3 (1:400, Affinity), rabbit anti-cleaved caspase9 (1:400, Affinity), rabbit anti-collagen I (1:700, Affinity), rabbit anti-α-smooth muscle actin (α-SMA; 1:1000, Abcam), rabbit anti-TOM20 (1:2000, Affinity), and mouse anti-BCL2L10 (1:400, Abcam). After incubating with fluorescence-conjugated secondary antibodies (1:20000, Rockland Biochemicals), the immunoreactive bands were visualized using an Odyssey Infrared Imaging System (LI-COR Biosciences). For the protein quantification, the bands were scanned and quantified using Image-Pro plus 6.0 software (Datacell, UK), and GAPDH served as an internal control.