Ecl chemiluminescent kit ecl plus

A549 cells were seeded on 6-well plates at a density of 9 × 10⁵ cells/well and cultured for 24 h. Then, cells were treated with different concentrations of ZIF-8 (0, 10, 25, 50, 75 and 100 µg/mL) for 24 h. At the end of the incubation period, the cells were lysed in RIPA lysis buffer (Beyotime Biotechnology, China) on ice for 15 min and protein was extracted. The lysate was centrifuged at 18,000× g for 5 min at 4 °C and the protein was collected. The protein concentrations of the samples were measured using a bicinchoninic acid assay (BCA) kit (Beyotime Biotechnology, China). Equal amounts (20 µg) of protein from the samples were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred to 0.22 µm PVDF membranes (Millipore, Boston, MA, USA). Proteins were then blocked for 1 h in 5% BSA and incubated overnight at 4 °C with primary antibodies of lysosomal-associated membrane protein 1 (LAMP1) and cathepsin B, followed by 1 h of incubation with HRP-conjugated secondary antibody. Target protein bands were visualized using ECL-chemiluminescent kit (ECL-plus, Thermo Scientific, Waltham, MA, USA). Proteins levels were semi-quantitatively analyzed using ImageJ software. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading protein. The relative expression levels of LAMP1 and cathepsin B were normalized to the level of GAPDH.

Liver tissues and HSCs were lysed in RIPA buffer for protein extraction by using centrifugal separation. Then we collected upper supernatant. After extraction, protein concentration was estimated by a BCA protein assay kit (Boster, China). Proteins extracted were loaded on to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose membranes (Millipore, Bedford, MA, United States). After blocked with 5% non-fat milk in TBST, the PVDF membranes were washed with TBST buffer at least for three times and cultured with special primary antibodies at 4°C overnight. Then the PVDF membranes were washed for three times with TBST buffer. Next, the membranes were incubated with corresponding secondary antibody for 1 h. Immunoreactive bands were detected with ECL-chemiluminescent kit (ECL-plus, Thermo Fisher Scientific). Image J software was used to quantify and analyze intensities of the bands. β-actin was developed as a loading control to verify the equal loading of proteins. Primary antibodies were as follows: ZEB2 (Santa Cruz Biotechnology, United States); TRAIL, Col. I, Caspase-3 (Bioss, Beijing, China), α-SMA (Sigma, United States), β-actin (Bioworld, United States).

Protein was extracted from AML-12 cells or liver tissues using RIPA lysis buffer (Beyotime, China) supplemented with 1% PMSF. After centrifugation for 30 min at 4°C at 12000 rpm, upper supernatant was collected. Protein was separated on 8–12% SDS-polyacrylamide mini-gels, then transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, United States). After blocked in 5% non-fat milk for 3 h, membranes were washed in TBS-Tween20 buffer three times per 10 min, and incubated with specific primary antibodies overnight. Primary antibodies Lipin1 (ab181389), PPAR-α (ab8934), SREBP-1 (ab28481), FASN (ab128856) were purchased from Abcam. Primary antibodies IL-6 (BS6419) was purchased from Bioworld. β-Actin (bs-0061R) and TNF-α (bs-10802R) were purchased from Bioss. Histone3 (17168-1-AP) was purchased from Proteintech. Then the membranes were washed for three times with TBS-Tween20 buffer, following incubation with the corresponding horseradish peroxidase conjugated secondary antibody for 1 h and then developed onto chemiluminescence. Western blotting detection system using ECL-chemiluminescent kit (ECL-plus, Thermo Scientific). Quantitative densitometric analyses of immunoblotting images were performed using ImageJ software. The experiment was repeated for three times.