Ab124734

Diphenyl dichloromethane, diallyl disulphide, hydrogen peroxide, 4-nitrophenol, methanesulfonic acid, tetramethoxypropane, dinitrophenylhydrazine, 1-methyl-2-phenylindole 4-nitrophenyl-N-acetyl-β-d-glucosaminide, acetonitrile, methanol, and guanidine were purchased from Sigma (St. Louis, MO, USA). The anti-angiotensin II type 1 receptor antibody (ab124734) and anti-angiotensin II type 2 receptor antibody (ab92445) were purchased from Abcam (Cambridge, MA, USA). The β-actin antibody GTX109639, eNOS antibody GTX54637, KEAP1 antibody GTX60660, and NRF2 antibody GTX103322 were purchased from GeneTex Inc. (Irvine, CA, USA). The nephrin antibody ALX-810-016-R100 was purchased from Enzo Life Sciences Inc. (Farmingdale, NY, USA). The catalase antibody sc-34281, heme oxygenase 1 antibody sc-1797, and superoxide dismutase antibodysc-8637 were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). All other chemicals used herein were of the highest analytical grade available.

Cells were homogenized in an ice-cold RIPA buffer (Keelton Co., Ltd., China). Protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Thermo, Rockford, United States). Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, Hercules, CA, United States) and transferred onto polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany). The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with 0.05% Tween 20 (TBST) at room temperature for 2 h, incubated overnight with the primary anti-CDK6 (1:1,000 dilution, ab241554; Abcam), anti-VEGF (1:2,000, GTX102643; GeneTex), antiAT1R (1:2,000, ab124734; Abcam), or anti-GAPDH antibodies (1:2,000 dilution, #5174;CST). After washing with TBST three times for 10 min, the membranes were treated with HRP-labeled secondary antibodies (1:1,000 dilution, Beyotime, China) for 1 h and visualized using the chemiluminescence method (Thermo, Rockford, United States). The relative protein expression was measured using GAPDH as an internal control.

NK/T-cell lymphoma samples and corresponding normal tissues were lysed in RIPA buffer. After electrophoresis and transfer to a nitrocellulose membrane, the proteins were probed with AT1R (1:2000, ab124734, Abcam, MA, U.S.A.), proliferating cell nuclear antigen (PCNA, 1:2000, ab19166, Abcam), Ki67(1:5000, ab92742, Abcam), p-PI3K (1:500, ab182651, Abcam), PI3K (1:1000, #4249, CST, MA, U.S.A.), p-Akt (1:2000, #4060, CST), Akt (1:1000, #4691, CST) primary antibodies, followed by incubation with secondary antibodies (Abcam, MA, U.S.A.). The bands were visualized using the enhanced chemiluminescence (ECL) substrate (BioChannel Biological Technology Co., Ltd.). The protein level was normalized to the protein level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:10000, ab18602, Abcam).

Total protein lysates were extracted from lung tissues or MLg2908 cells by homogenization in the Laemmli sample buffer. An equal amount of proteins (30 μg per lane) were separated by electrophoresis on 8% polyacrylamide gels, and the proteins were electro-transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA) overnight. The membranes were then incubated with primary antibodies as follows: anti-β-actin (1:1000, Santa Cruz), anti-α-smooth muscle actin (SMA) (1:1000, CBL171, Millipore), anti-transforming growth factor (TGF)-β1 (1:1000, ab92486, Abcam), anti-collagen type 1 (Col I) (1:1000, ab21286, Abcam), anti-fibronectin (FN) (1:1000, F7387, Sigma-Aldrich), anti-vitamin D receptor (VDR) (1:1000, sc-13133, Santa Cruz), anti-renin (1:1000, sc-133145, Santa Cruz), anti-AT1R (1:1000, ab124734, Abcam) and anti-AGT (1:1000, sc-374511, Santa Cruz). Secondary antibody was horseradish peroxidase-conjugated anti-IgG (ZB-2301, ZB-2305, ZSGB-BIO). The relative amount of proteins in each band was quantified using ImageJ (NIH), and normalized to β-actin (TA-09, ZSGB-BIO) as an internal loading control.

DOX was purchased from Zhejiang Hisun Pharmaceutica Co, Ltd. (Taizhou, China); Val and Diphenyleneiodonium chloride (DPI) were purchased from Meilunbio Inc (Dalian, China); MTT reagent and Apoptosis Detection Kit were purchased from Sigma-Aldrich Inc (WI, USA); NOX2 siRNAs, NOX4 siRNAs, negative siRNA, NOX2 plasmid, NOX4 plasmid and empty vector were constructed by GenePharma Inc (Shanghai, China); Lipofectamine 2000 and Protein marker (PageRuler™) were purchased from Invitrogen (CA, USA); Trizol reagents and PCR kit were purchased from Takara Bio Inc (Dalian, China); The primer sequence of NOX2, NOX4 and GAPDH were synthesized by ShineGene Inc (Shanghai, China); Primary monoclonal antibodies for Angiotensin II Type 1 Receptor (AT1R) (ab124734), NOX2/gp91phox (ab129068), NOX4 (ab79971), caspase-3 (ab184787), caspase-9 (ab184786), p-p38 (ab4822), p38 (ab170099), p-JNK (ab124956), JNK (ab179461), p-ERK (ab201015), ERK (ab184699), β-actin (ab8227) and secondary antibodies (ab6721) were purchased from Abcam (Cambridge, UK).

Antibodies against AGTR1 (catalog ab124734, 1:1000), AGTR2 (catalog ab92445, 1:1000), eNOS (catalog ab199956, 1:1000), p-eNOS (catalog ab215717, 1:1000), and HIF1α (catalog ab179483, 1:1000) were bought from Abcam. Antibodies against β-actin (catalog ap0060, 1:2000) were purchased from Bioworld. Reactive oxygen assay kits were purchased from Biyuntian. TBARs assay kits were purchased from Cayman. The nitrate oxide/nitrite (NOx) assay kits were purchased from Cayman. L-NAME was purchased from MCE. FG-4592 (Roxadustat), the HIF prolyl hydroxylase inhibitor, was purchased from Selleck Chemicals.