Ab13533

Partial kidney tissues were thoroughly homogenized in a lysis buffer (0.97% protease inhibitor cocktail, 0.94% 50 mM phenylmethylsulfonyl fluoride, and 97.09% 1x RIPA) on ice. The protein concentrations of the homogenates were measured using the BCA Protein Assay Kit (Merck Millipore, USA); 50 μg of protein was electrophoresed on 12% SDS-PAGE, transferred onto polyvinylidene difluoride (PVDF) membrane (Merck Millipore, USA), and blocked in 5% bovine serum albumin (BSA) in Tris-buffered saline. Then, the bands were incubated overnight at 4°C in a corresponding primary antibody solution containing Nrf2 (ab137550), HO-1 (ab13248), SOD1 (ab13498), SOD2 (ab13533), and CAT (ab16731) (1 : 2000; Abcam, UK) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH, ABS16, 1 : 2000, Merck Millipore, USA) and then incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (bs-0295G, 1 : 2000, Beijing Biosynthesis Biotechnology Co. Ltd., China) for 4 hours at 4°C. Specific signals were visualized with ECL detection on a gel imaging system (UVP, California, USA). The average optical density of the bands was quantified using ImageJ (National Institutes of Health, Bethesda, USA).

Western blot analysis was performed as described previously^{8 (link)}. In brief, whole kidney homogenates were lysed in the presence of protease inhibitors. Clear protein extracts were obtained by centrifugation at 12,000 g for 10 min. 20 μg proteins were transferred to subjected to 4%-12% gradient SDS-PAGE, transferred onto a PVDF membrane filters (Bio Rad). PVDF membranes were blocked with 5% dry milk for 1 h. Membranes were incubated in the primary overnight at 4 °C. The following antibodies used in the study are: anti-PRR (1:1000, HPA003156, Sigma), anti-NOX4 (1:1000, ab133303, Abcam), anti-SOD2 (1:1000, ab13533, Abcam), anti-UCP2 (1:1000, ab203244, Abcam), anti-Caspase 3 (1:1000, CST #9665, Cell signaling), anti-pNF-κB Ser536 (1:1000, ab86299, Abcam), anti- tNF-κB (1:1000, ab16502, Abcam), anti-collagen IV (1:1000, ab6586; Abcam), anti-fibronectin (1:1000, ab2413; Abcam), anti-sirt1 (1:200, sc-15404, Santa Cruz), anti-pFOXO3a ser318/312 (1:1000, CST #9465, Cell signaling), anit-FOXO3a (1:200, sc-48348, Santa Cruz), and anti-β-actin (1:5000, sc-47778, Santa Cruz). This was followed by incubation with the corresponding secondary antibody (horseradish peroxidase-labeled IgG, 1:1000). β-actin was used to normalize per total amount of loaded proteins. Chemiluminescence blot images were captured by the UVP imaging and analyzed by using Image J software (NIH, Bethesda, MD).

For whole cell extraction, the B4G12 cells were lysed with RIPA buffer (Beyotime, P0013B, Beijing, China). The detailed procedure has been previously described [9 (link)]. The primary antibodies were used as following: α-tubulin (11224-1, Proteintech), p16 (ab108349, Abcam), p21 (ab109520, Abcam) and SOD2 (ab13533, Abcam).

After boiling in Laemmli′s sample buffer, proteins (40 μg) were resolved on 7%, 12% or 15% SDS-polyacrylamide gels, and transferred to the PVDF membrane. Membranes were blocked with 5% BSA, and incubated with primary antibody. CTR1, DMT1, ATOX1, COX17, COX2 and CCS were detected using Santa Cruz Biotechnology (Santa Cruz, CA, USA) antibodies: sc-18473, sc-166884, sc-398742, sc-393617, sc-514489, sc-55561, respectively. SOD1, SOD2, CAT, GR and GPX, were detected using Abcam (Trumpington, Cambridge, UK) antibodies: ab13498, ab13533, ab16731, ab16801, ab22604, respectively. Anti-MT-1/MT-2 antibody (M0639) was the product of Dako (Agilent, Santa Clara, CA, USA). β-actin was detected by AC-15 antibody (Sigma-Aldrich, St. Louis, MO, USA). The blots were incubated with anti-rabbit, anti-goat, or anti-mouse secondary antibodies conjugated with horseradish peroxidase. Immunoreactive proteins were visualized by the enhanced chemifluorescence method. Quantitative analysis of immunoreactive bands was done using iBright^TM FL1500 Imaging System Software (Thermo Fisher Scientific, Waltham, MA, USA). All experimental samples and controls used for one comparative analysis are run on the same blot/gel.

JWH133, a selective CB2 agonist, was purchased from Tocris Bioscience (Space Import-Export Srl, Milano, Italy). AM630, a selective antagonist, was purchased from Tocris Bioscience. Furthermore, we purchased rabbit anti-rat iNOS (ab15323), SOD2 (ab13533), NOX4 (ab154244), TNF-α (ab6671), IL-1β (ab9722), IL-6 (ab6672), Collagen II (ab34712), Aggrecan (ab3778), MMP3 (ab53015), and MMP13 (ab39012) antibodies from Abcam (Cambridge UK). Secondary antibodies were purchased from Wuhan Amictech Technology Co Ltd. Phosphate-buffered saline (PBS) was purchased from Beyotime (Beyotime Institute of Biotechnology, Jiangsu, China). ROS production was detected by 2′,7′-dichlorodihydrofluorescein diacetate fluorescent probe purchased from Beyotime as well. Fetal bovine serum, Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 (DMEM/F12), RIPA protein lysates. Trizol reagent and a BCA protein kit were purchased from Invitrogen (Waltham, MA, USA). Type II collagenase was purchased from Thermo Fisher Scientific. Primary antibodies (Western Blot) were purchased from Abcam and secondary antibodies were purchased from Beijing Zhongshan Jinqiao Biotechnology. Triton-X-100 was purchased from Invitrogen and DAPI was purchased from Beyotime.

Protein samples were resolved by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis for 2 h and electrophoretically transmitted to a PVDF membrane (Merck Millipore, USA). After blocking nonspecific binding sites with 5% skim milk for 60 min at room temperature, the membranes were incubated overnight at 4°C with primary antibodies against SIRT3 (1 : 1000; ab189860), Bax (1 : 1000; ab32503), Bcl-2 (1 : 1000; ab59348), ALP (1 : 1000; ab95462), Osterix (1 : 500; ab22552), OCN (1 : 500; ab93876), β-actin (1 : 1000; ab8227), SOD2 (1 : 5000; ab13533), and Ac-SOD2 (1 : 5000; ab137037) (all from Abcam, Cambridge, UK). Then, the membranes were rinsed in Tris-buffered saline with Tween 20 and incubated with the corresponding secondary horseradish peroxidase-conjugated antibodies (1 : 1000) for 2 h at room temperature. The proteins were detected using chemiluminescent HRP substrates (Millipore Corporation, Billerica, MA, USA).