Tris buffered saline tween

DHA and Brusatol (Nrf2 inhibitor) were purchased from Tauto. GSH assay kits and antibodies against β‐actin were purchased from Beyotime. The hydroxyproline kit was purchased from the Jiancheng Bioengineering Institute. The TNF‐α and TGF‐β ELISA kits were purchased from Elabscience. Antibodies against HO‐1 were purchased from Abcam. Antibodies against tumor necrosis factor α (TNF‐α), transforming growth factor‐β (TGF‐β), GPX4, Nrf2, electron microscope fixative, hydrogen peroxide (H₂O₂), hematoxylin, eosin, osmium acid, lead citrate, uranium acetate and the immunohistochemistry (IHC) special goat anti‐rabbit secondary antibody were purchased from Servicebio. The goat anti‐rabbit IgG (H + L) fluorescent secondary antibody was purchased from Cell Signaling Technology; excitation is 777 nm and peak fluorescence emission is 794 nm. The diaminobenzidine (DAB), sodium dodecyl sulfate (SDS), radioimmunoprecipitation assay (RIPA) buffer and Tris Buffered Saline Tween (TBST) were purchased from Solarbio. The polyvinylidene fluoride membrane was purchased from Millipore. The acetone and ethanol were purchased from Chron Chemicals.

The expression of E1A protein, Hexon protein, and apoptosis-related proteins (p53, Bcl-2, Bax, cleaved caspase-3, and caspase-3) were detected by Western blot analysis. The total proteins were extracted using RIPA with phenylmethanesulfonyl fluoride (PMSF) (Beyotime Biotechnology, Jiangsu, China), and quantized using a BCA Protein Assay Kit (Beyotime). Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) was carried out to separate the total protein. The separated protein was transferred to a PVDF membrane (Millipore, Bioprocess Technology Center, Billerica, MA, USA), blocked with skimmed milk powder, and incubated in the first antibodies overnight at 4 °C. The membranes were incubated in horseradish peroxidase -conjugated secondary antibodies for 2 h. After extensive washing with 20 ml Tris-buffered saline-Tween (TBST; Solarbio), the proteinwas visualized at Fast Chemiluminescence Image System (ImageQuant 350, GE Healthcare Life Sciences, UAS). The housekeeping genes β-actin was viewed as internal reference.

As previously described [14 (link)], total protein from each sample was extracted with RIPA lysis buffer (Sigma, St, Lousis) containing 1 mmol/L phenylmethylsulfonyl fluoride (PMSF, Solarbio). A phosphorylase inhibitor and a protease inhibitor cocktail (1:100, Solarbio) were added to prevent degrading of proteins in the extracts. tThe protein concentration was quantified using a BCA Protein Assay Kit (Beyotime, China). Afterwards, the equal boiled protein samples (30 μg) were loaded on 8 ~ 12% sodium dodecyl sulfatepolyacrylamide gels (Solarbio Life Sciences), electrophoretically separated, and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, Massachusetts), which had been wetted in 100% methanol for 15 s previously.. Then the membranes were blocked in 5% skim milk containing Tris-buffered Saline Tween (TBST, Solarbio, China) at room temperature for 1 h to reduce nonspecific bindings. Subsequently, the membranes were incubated with the primary antibodies (1:500 ~ 1:1000 dilutions, Cell Signaling Technology, USA) overnight at 4 °C with gentle agitation. Getting washed 2 ~ 3 times with TBST, the secondary antibodies (1:2000 dilution) were added for incubation for 1 h. Finally, the immunoreactive bands were detected with Chemiluminescent HRP Substrate (Merck Millipore) and quantified through a Image Lab software (Bio-Rad, Hercules, USA) .

After washing twice with ice-cold phosphate-buffered saline (PBS), total protein was extracted from treated cells with a mixture of RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China), benzenesulfonyl fluoride (PMSF) (Beyotime Biotechnology), and a protein phosphatase inhibitor (Sigma). The protein concentration was determined via the BCA method after centrifugation at 12,000 rpm for 10 min. Protein was separated on a 10% SDS polyacrylamide gel, transferred to nitrocellulose membranes, and blocked in a blocking solution at room temperature for 1 h. The primary antibodies used were as follows: anti-HIF-1α (#36169), anti-Akt (#2920), anti-p-Akt (Ser473) (#4060), anti-mTOR (#2983), anti-p-mTOR (Ser2448) (#5536), and anti-β-actin (#4970S). All the primary antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). After incubation with primary antibodies at 4°C overnight, the membranes were washed three times with Tris-buffered saline-Tween (TBST) (Solarbio Biotechnology) and then incubated with a horseradish peroxidase-conjugated secondary antibody (Zhongshan Golden Bridge Biotech; dilution 1 : 5,000) for 1 h at room temperature. Protein bands were visualized with an enhanced chemiluminescence kit (Thermo Fisher Scientific, Suwanee, GA, USA). The band intensity of target proteins was normalized to that of β-actin.

Total proteins were extracted from PBMCs using RIPA lysis buffer (Solarbio, China), and protein concentration was determined using the Pierce™ BCA Protein Assay Kit (Thermo Scientific, USA). An equal amount of protein was loaded onto 10% SDS-PAGE and subjected to electrophoresis, then transferred onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad, USA). The membrane was washed with Tris-buffered saline-Tween (TBST) (Solarbio, China), and blocked with 5% skim milk powder or bovine serum albumin (BSA) (Sigma-Aldrich, Germany). Incubation was carried out using specific primary antibodies against STAT3 (Abcam, U.K), pSTAT3 Y705 (Abcam, U.K), ADAM17 (R&D Systems, USA), IL-23 R (Abcam, U.K), and GAPDH (Santa Cruz, USA), followed by the incubation with the secondary antibodies HRP Goat anti-Mouse antibody (abclonal, USA) or HRP Goat Anti-Rabbit IgG (abclonal, USA). Pierce™ ECL Western Blotting Substrate (Thermo Fisher, USA) was used as the chemiluminescent substrate, and X-ray imaging was used to image the bands. Relative expression levels of proteins were quantified by densitometric values of fluorogram bands which was analyzed by Image J software (NIH, USA) and each value was normalized to those corresponding control protein band.

These cells were lysed using RIPA buffer (Solarbio, China) for 30 min on ice, and then centrifuged at 4°C at 1 2000 rpm for 10 min to collect total protein. The extracted protein was quantified through the Pierce™ BCA Protein Assay Kit (ThermoScientific, USA). The protein was separated using a 12% SDS-polyacrylamide gels electrophoresis (SDS-PAGE), and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). The PVDF membrane containing protein was blocked using 5% skim milk at a room temperature for 2 h. Next, STAT3, pSTAT3 Y705, NF-κB p65, ERK, pERK, and GAPDH (antibodies were purchased from Abcam, UK) specific primary antibodies were added and incubated overnight at 4°C. After incubating with primary antibodies, the PVDF membrane was washed using Tris-buffered saline-Tween (TBST, Solarbio, China), and incubated at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibody (goat anti-rabbit or anti-mouse IgG, Santa Cruz, USA) for 1 h. The immunoreactive bands were visualized using enhanced chemiluminescence kit (Millipore, USA), and protein bands were quantitatively analyzed through the Image J software (NIH, USA).