Ripa buffer

Bladder cancer cells were washed with phosphate-buffered saline and lysed in RIPA buffer (cwbiotech) containing protease inhibitor. The protein concentration was quantified using the BCA Protein Assay Kit (cwbiotech). For each sample, an amount of 20 μg total protein was separated by 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes for 1 h and a half at 320 mA in transfer buffer. After blocking with 5% degreased milk at room temperature for 1 h, the membranes were incubated overnight at 4 °C with rabbit polyclonal anti-human LBHD1 (1:1000, Abcam) and mouse monoclonal anti-GAPDH (1: 2,000, cwbiotech). After washing, the membranes were incubated with the corresponding secondary antibodies conjugated with horseradish peroxidase for 1 h at room temperature. Immunoreactive bands were incubated with ECL western blotting detection reagents (Amersham Biosciences) then visualized using a western blotting detection system (ECL Plus; Amersham Pharmacia Biotech, Little Chalfont, UK). The film was scanned, and the intensity of the bands was quantified by densitometry (Image J 1.47 software) and normalized to the corresponding GAPDH bands. All of the Western immunoblots were performed at least three times.

For western blot analysis, RIPA buffer (CWBIO, Beijing, China) was employed to generate cell lysates. Using a bicinchoninic acid protein assay kit, protein concentrations were measured (Beyotime Biotechnology, China). Using SDS-PAGE, 30 μg of each protein sample was separated and then transferred to PVDF membranes (Millipore). The membranes were then incubated overnight at 4°C with the primary antibody (anti-MEX3C from Abcam or anti-β-actin from Cell Signaling Technology). The next day, membranes were incubated with HRP-conjugated secondary antibodies at room temperature for one hour. Millipore chemiluminescence detection reagents were used to identify signals. In this study, β-actin was utilized as a loading control.

Immunoblotting was performed as a previous report (Zhang et al., 2021 (link)). Cells were lysed by using RIPA Buffer (CWBio, China) containing protease inhibitors to extract total protein. Total protein (20 μg) was added to 10% SDS-polyacrylamide gel. The protein was transferred to PVDF membranes (Millipore, USA) after electrophoresis and blocked for 1 hr with a blocking buffer (Beyotime, China). Then PVDF membranes were incubated with the primary antibodies including ALP (Abcam, UK, 1:3000), RUNX2 (CST, USA, 1:2,000), S1PR1 (Abcam, UK, 1:2,000), and GAPDH (CST, USA, 1:5,000) overnight at 4°C. The membranes were further incubated with horseradish peroxidase-conjugated secondary antibody for 1 hr and reacted with ECL (Millipore, USA). Finally, the photographs were taken by Tanon-5200 system (Tanon, China). The densitometry of protein bands was quantified by ImageJ 1.51K.

Proteins were extracted using RIPA buffer (CWBIO, China). The protein concentration was measured using a BCA assay kit. The samples were mixed with SDS-PAGE loading buffer, electrophoresed via SDS-PAGE, and transferred onto Millipore or GE PVDF membranes. The PVDF membranes were blocked with 5% skim milk for 1 h and incubated with primary followed by secondary antibodies (all 1:5,000, Affinity, China). The membranes were then bound to ECL and analyzed using an image system software (Protein Simple, USA).
The following primary antibodies were employed: anti-p65 (1:1,000, CST #6959), anti-p-p65 (1:1,000, ab194726), anti-LC3 (1:2,000, ab192890), anti-ADPN (1:1,000, ab22554), anti-HMGB1 (1:1,000, ab79823), anti-GPX4 (1:1,000, ab125066), anti-P62 (1:1,000, ab109012), anti-YAP (1:1,000, CST#8418S), anti-STAT3 (1:1,000, ab109085), and anti-β-actin (1:5,000, Sigma).

LMH cells infected with the recombinant virus were collected and lysed with RIPA buffer (CWbio, Beijing, China) containing protease inhibitors and phosphatase inhibitors (CST, MA, USA). The lysates were boiled for 10 min and centrifuged for 3 min. After SDS-PAGE, the proteins were transferred onto nitrocellulose membrane (GE Healthcare Life sciences, Freiburg, Germany). After blocked with 5% skim milk in PBST for 2 h at room temperature (RT), the membrane was incubated with mAb 5C3 for 2 h at RT. After three washes with PBST, the membrane was incubated with HRP-conjugated goat anti-mouse IgG (Sigma-Aldrich, USA) for 1 h. The dilution ratio of the secondary antibody was 1:10000. After another three washes, the membrane was developed with chemiluminescent reagents and imaged with an automatic imaging system (Tanon 5200).

The rat lung tissues and cell samples were scraped and resuspended in a 0.5 mL RIPA buffer (CWBIO, Jiangsu, China). Tissue or cell lysates were placed on ice for 1 h. After centrifugation at 10,000× g for 20 min, the protein concentration was determined using the BCA kit (Beyotime, Shanghai, China). Then, 20 μg samples were electrophoresed via 10% SDS-PAGE (Yeasen Biotechnology, Shanghai, China) and transferred onto a PVDF membrane. Samples were blocked with 5% nonfat milk in TBST solution at room temperature for 2 h and then probed with primary antibodies p38 (CST, Boston, USA, dilution: 1:1000), p-p38 (CST, Boston, USA, dilution: 1:1000), Smad3 (CST, USA, dilution: 1:1000), p-Smad3 (CST, Boston, USA, dilution: 1:1000), GAPDH (Utibody, Tianjin, China, dilution: 1:2000) at 4 °C overnight. Secondary antibodies used for detection included HRP-conjugated antirabbit IgG and antimouse IgG (Bioss, Beijing, China, dilution: 1:3000), and the protein expression was detected using an ECL chemiluminescence detection kit (Boster, Wuhan, China).