Chemiscope western blot imaging system

Cells were lysed with radio-immunoprecipitation assay lysis buffer (APExBIO, K1020), supplemented with protease inhibitor cocktail (APExBIO, K1007) and phosphatase inhibitor cocktail (APExBIO, K1015). Insoluble cell debris was removed by 15-min centrifugation at 13,500×g at 4 °C. Protein concentration was determined using a bicinchoninic acid protein assay kit (Beyotime, P0010S). Western blot was performed on total protein extracts. Proteins were then transferred to 0.45-μm polyvinylidene difluoride membranes (Sigma, IPVH00010) after electrophoresis. Membranes were blocked for 1 h at room temperature in blocking buffer (Beyotime, P0023B) and incubated overnight at 4 °C with antibodies in primary antibody dilution buffer (Beyotime, P0023A). Goat-anti-Rabbit horseradish peroxidase-conjugated (Abcam, ab2057181:10,000) antibody was used as secondary antibody. Protein bands were visualized with Clarity Western ECL Substrate (Bio-Rad, USA) and the pictures were obtained by ChemiScope Western Blot Imaging System (Clinx Science Instruments, China).
Primary antibodies and corresponding concentrations were used as follows: rabbit monoclonal anti-SUN1 (Abcam, ab124770, 1:1000) and rabbit monoclonal anti-SUN2 (Abcam, ab124916, 1:1000). Glyceraldehyde 3-phosphate dehydrogenase (Abcam, ab8245, 1:1000) was used as a loading control.

The culture supernatants were collected and concentrated as previously described (Zhao et al., 2021 (link)). Cell pellets were washed in ice-cold PBS and lysed with RIPA containing the protease inhibitor of phenylmethanesulfonyl fluoride (PMSF, 1 mM). The protein samples were quantified with a BCA Protein Assay Kit (Thermo Scientific, USA) according to the manufacturer’s protocol. Each sample (30 μg) of cell lysates or cell supernatants (10 μl) were separated by 12% SDS-PAGE, and the electrophoresis protocol was set as follows: 80 V 1 h 05 min and 120 V 30 min. Then, the protein was transferred onto 0.22 or 0.45 μm PVDF membrane (Millipore, USA) under conditions of 200 mA/1 h or 1 h 30 min at 4°C. The membranes were blocked in 5% bovine serum albumin (BSA) in PBST for 2 h at room temperature and incubated at 4°C overnight with different primary antibodies (Abs), including IL-1β (1:1,000), caspase-1 (1:1,000), NLRP3 (1:1,000), and β-actin (1:5,000). The HRP-conjugated secondary antibodies were rabbit anti-goat IgG (H+L) (1:5,000) or goat anti-mouse IgG (H+L) (1:5,000, EarthOx, USA). The blots were developed using the ChemiScope Western Blot Imaging System (Clinx Science Instruments, China).

The day before assay, RAW264.7 macrophages (5.0 × 10⁵ cells/well) were seeded in 6-well plates. Cells were left untreated or exposed to 1.25, 2.5 nM CdSe/ZnS QDs for 48 h. The medium was removed and cells were washed three times with PBS. Protein was extracted by the cold mammalian protein extraction reagent (Thermo, USA) according to the manufacturer’s instructions. A total of 15 μg proteins were resolved by 12 % SDS-PAGE and transferred to 0.22 μm polyvinylidene difluoride membranes (Millipore, USA). Membranes were soaked in blocking buffer (Skim Milk, China) for 1 h at room temperature and incubated with rabbit antibodies of GAPDH (1:1000, ABclonal, USA), Caspase-9 (1:1000, Proteintech, China), Caspase-3 (1:1000, Proteintech, China) at 4 °C overnight. Membranes were washed with TBST (10 mM Tris–HCL, 150 mM NaCl, and 0.05 % Tween 20) three times at 10 min interval. Membranes were incubated with goat anti-rabbit secondary antibodies (1:1000, ABclonal, USA) for 1 h. Then the membranes were washed three times with TBST. Protein bands were visualized with clarity western ECL substrate (BioRad, USA) and the pictures were obtained by chemiscope western blot imaging system (Clinx science Instruments, China).

Liver samples or hepatocyes were lysed with RIPA peptide lysis buffer (Beyotime Biotechnology, Jiangsu, China) containing 1% protease inhibitors (Pierce) and Western blotting analysis were performed according to standard procedures. Primary antibodies were used as follows: anti‐Pknox1 (1:1000, Novus, USA), anti‐IR (1:1000; Abcam, Cambridge, UK), anti‐pY‐IRS1 (1:1000; Abcam), anti‐IRS1 (1:2000; Abcam), anti‐pS‐IRS1 (1:2000; Abcam), anti‐pY‐IRS1 (1:1000; Abcam), anti‐AKT (1:1000; Cell Signaling Technology, MA, USA), anti‐pY‐AKT (1:1000; Cell Signaling Technology), and anti‐β‐actin (1:3000, Huabio, China). Protein bands were developed using the Enhanced Chemiluminescence (ECL) system and were visualized by using the ChemiScope Western Blot Imaging System (Clinx Science Instruments Co., Ltd). The gray‐scale value assay was performed by using Image J software (Rawak Software, Inc. Germany).

HCC samples or HCC cells were lysed with RIPA peptide lysis buffer (Beyotime Biotechnology, Jiangsu, China) containing 1 % protease inhibitors (Pierce) and Western blotting analysis were performed according to standard procedures. Primary antibodies were used as follows: anti-SPAG9 (1:1000, Abcam), anti-JNK, anti-p-JNK, anti-c-Jun, and anti-MMP9 (1:2000, Abcam) and anti-GAPDH (1:3000, Huabio). Protein bands were developed using the Enhanced Chemiluminescence (ECL) system and were visualized and quantified by using the ChemiScope Western Blot Imaging System (Clinx Science Instruments Co., Ltd).

After treatment with AMSC-Exo or transfection with the mTOR expression plasmid, HCC cells or tumor samples were lysed with RIPA peptide lysis buffer (Beyotime Biotechnology, Jiangsu, China) containing 1% protease inhibitors (Pierce). The protein content of different fractions was determined via the BCA method. Equivalent amounts of protein (20 μg) were separated by SDS-PAGE with 10% gels and then were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA) and blocked with 1% BSA in TBST for 1 h at room temperature. The membranes were incubated with mTOR and phosphorylated-4EBP1 and -70S6K or GAPDH (Abcam) antibodies overnight at 4 °C. After washing, the membrane was incubated with an HRP-conjugated secondary antibody (1:3000; Abcam) for 1 h. Protein bands were identified using an enhanced chemiluminescence system and were visualized using a the ChemiScope Western Blot Imaging System (Clinx Science Instruments Co., Ltd). The gray value assay was performed by using ImageJ software (Rawak Software, Inc. Germany).