Hrp conjugated goat anti rabbit antibody

Cells were harvested and lysed on ice with RIPA buffer (Beyotime, China). Next, the lysate was incubated on ice for 15 min and then centrifuged for 15 min (13,000 rpm, 4 °C). The supernatant was collected, and the protein concentration was determined by using the BCA Protein Assay Kit (Beyotime, China). Protein samples (20 μg) were loaded into each lane of the SDS-PAGE lane. Following electrophoresis, proteins were transferred to PVDF membranes blocked with 3% BSA. Protein transferred membranes were incubated with primary antibodies overnight at 4 °C, followed by 1 h of incubation with secondary antibodies at room temperature. Western blots were visualized on a BioSpectrum 515 (Wolf Laboratories Limited, UK) by using the Affinity ECL kit (Affinity, china). The antibodies used in the western blot were: FN1 (1:1000 dilution, Abcam, USA), SRSF3 (1:300 dilution, Santa Cruz Biotechnology, USA), GAPDH (1:5000 dilution, Abcam, USA), HRP-conjugated goat anti-mouse (1:5000 dilution. Beyotime, China), and HRP-conjugated goat anti-rabbit antibody (1:5000 dilution, Beyotime, China).

Four human HCC and one normal liver cell lines were used in this study. SMMC-7721 and L02 cells were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). Huh7 cell was from Riken Cell Bank (Tsukuba Science City, Japan). MHCC-97H and MHCC-97L cells were from Liver Cancer Institute, Zhongshan Hospital of Fudan University (Shanghai, China) [11] (link). SMMC-7721 and L02 cells were cultured in RPMI medium 1640 (GIBCO, USA). Huh7, MHCC-97H and MHCC-97L cells were cultured in DMEM supplemented with 10% fetal bovine serum (GIBCO, Austria). Cells were cultured in 37°C incubator with humidified atmosphere containing 5% CO₂. Goat anti-human Nova1 polyclonal antibody was purchased from LifeSpan Biosciences (Seattle, USA) for detecting Nova1 in HCC tissues and from Abcam (Cambridge, USA) for detecting Nova1 in cell lines. HRP-conjugated rabbit anti-goat antibody was purchased from EarthOx, LLC (San Francisco, USA). Mouse anti-GAPDH antibody, HRP-conjugated goat anti-mouse antibody and HRP-conjugated goat anti-rabbit antibody were purchased from Beyotime Institute of Biotechnology (Shanghai, China). The enhanced chemiluminescence Western blotting Substrate System was purchased from Pierce (Rockford, USA).

Monoammonium glycyrrhizinate (MAG) was purchased from Sigma Chemical Co. (St. Louis, MO, USA) and suspended in phosphate-buffered saline (PBS). Lipopolysaccharide (Escherichia coli 055:B5) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Mouse TNF-α and IL-1β enzyme linked immunosorbent assay (ELISA) kits were purchased from R&D (Minneapolis, MN, USA). Mouse mAb NF-κB p65 and mouse mAb IκBα were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). HRP-conjugated goat anti-rabbit antibody was provided by Beyotime (Haimen, China). The Nuclear and Cytoplasmic Protein Extraction Kit and BCA protein assay kit were provided by Thermo Scientific (Rockford, IL, USA).

The TG tissues were cut into pieces and homogenised with the RIPA lysis buffer plus phenylmethanesulfonyl fluoride (PMSF) at a ratio of 20 mg to 150–250 μL, resulting in a final concentration of 1 mm. The homogenate was incubated on ice for 30 min followed by centrifugation at 14 000 × g for 5 min. The supernatant was stored at −80 °C before analysis. The lysates were run on SDS-PAGE and separated by electrophoresis (Tanon, Shanghai, China). Proteins were transferred onto polyvinylidene fluoride (PVDF) membranes and blocked with 5% skim milk in a Tris-buffered saline with Tween 20 (TBST) solution for 1 h at room temperature. The sealed PVDF membranes were incubated in the primary TRPV1 goat polyclonal antibody (1:200, Santa Cruz, USA) for 2 h at room temperature, washed with TBST four times and incubated with the second HRP-conjugated goat anti-rabbit antibody (Beyotime Biotechnology, China) for 1 h. The protein blot densities were analysed using ImageJ Software (National Institutes of Health, Bethesda), which marked the target intensity band as A_TRPV1 and the internal reference intensity band (GAPDH) as A_GAPDH; protein expression at the corresponding time point = A_TRPV1/A_GAPDH.

200 μL of rAAV samples at a concentration of 1 × 10¹⁰ vg/mL were added to 0.2 g of HSPG medium (Cytiva, #17-0407-01). After vortexing the mixture every ten minutes, it was allowed to stand at room temperature for thirty minutes. After 30 minutes of incubation with the above complexes, the PVDF membrane (Millipore, #IEVH85R) was rinsed twice with 0.5% PBST for five minutes each. The membrane was then incubated with a 5% skim milk solution for 60 minutes at room temperature and then incubated overnight at 4 °C with HSPG2-specific polyclonal antibody (Proteintech, #19675-1-AP). The membrane was then incubated with HRP-conjugated goat anti-rabbit antibody (Beyotime, #A0208, 1:5000) for 60 minutes at room temperature. The PVDF membrane was then recoated with 0.5% PBST three times for 5 minutes each. Finally, a Bio-Rad gel imager was used to visualize the membrane.

Autophagic activity of the cells was evaluated by LC3 (microtubule-associated protein 1 light chain 3) detection with immunoblotting after treatment with 50 nmol/L rapamycin (Sigma, CA, USA) for 2 h. Total cell proteins were extracted with RIPA buffer (Beyotime, Suzhou, China). Protein concentration was determined by BCA assay (Beyotime). Protein exacts were separated using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Beyotime) and transferred onto ployvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). Then, the membrane was blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 30 min at room temperature. After incubation with rabbit anti-rat LC3 antibody (1:500, Novus, Littleton, CO, USA) and mouse anti-rat β-actin antibody (1:3000, EMD Millipore) at 4 °C overnight respectively, the membrane was incubated with HRP-conjugated goat anti-rabbit antibody (1:5000) and HRP-conjugated goat anti-mouse antibody (1:1000; Beyotime) at room temperature for 2 h. After washing in TBST for three times, the blots was detected by BeyoECL (Beyotime) and imaged in ChemiDoc XRS+ system (Bio-Rad Laboratories, Hercules, CA, USA). Relative expression of LC3-II was shown as a ratio of LC3-II/β-actin.