HEK293T and HeLa cells were seeded separately into 100 mm plates. HEK293T cells were co-transfected with ADAR1 or ADAR2 and 5HT2C expression vectors as described above. Twenty-four hours after transfecting HEK293T cells or seeding HeLa cells, cells were washed with 1× DPBS and incubated with high-glucose DMEM containing 3.7 or 0 g/l NaHCO3 for 24 h. HEK293T and HeLa whole cell lysates were prepared using RIPA lysis and extraction buffer. 20 μg of each sample was resolved by electrophoresis on a 4–20% gradient SDS-PAGE gel and then transferred to a nitrocellulose membrane using a Trans-Blot® SD semi-dry transfer cell (Bio-Rad). After blocking the membrane with Intercept PBS blocking buffer (LI-COR) for 1 h, the membrane was probed using either a mouse ADAR1 monoclonal antibody (sc-73408, Santa Cruz; 1:1000 dilution) or an affinity-purified rat ADAR2 antiserum (Exalpha Biologicals; 1:375 dilution), as well as a polyclonal affinity-purified β-actin antibody (sc-1616-R, Santa Cruz; 1:1000 dilution). ADAR1 and β-actin signals were detected using IRDye secondary antibodies (LI-COR; 1:20,000 dilution), and ADAR2 signals were detected using Alexa Fluor® 790 light-chain specific anti-sheep IgG secondary antibody (Jackson ImmunoResearch; 1:50,000). All blots were imaged using an Odyssey CLx infrared imaging system (LI-COR) and quantified using Image Studio Lite (LI-COR).
Sc 73408
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-73408 is a laboratory product manufactured by Santa Cruz Biotechnology. It is an analytical instrument used for scientific research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Ab231303, by Abcam (3 mentions)
Np 40, by Merck Group (2 mentions)
Nupage sds page sample loading buffer, by Thermo Fisher Scientific (2 mentions)
Hrp coupled secondary antibodies, by GE Healthcare (2 mentions)
Complete protease inhibitor, by Roche (2 mentions)
Ab51841, by Abcam (2 mentions)
Horseradish peroxidase conjugated anti mouse antibody, by Santa Cruz Biotechnology (2 mentions)
Cellytic mt, by Merck Group (2 mentions)
Sc 1616, by Santa Cruz Biotechnology (2 mentions)
Ab92536, by Abcam (2 mentions)
Intercept pbs blocking buffer, by LI COR (1 mentions)
Odyssey clx infrared imaging system, by LI COR (1 mentions)
Image studio lite, by LI COR (1 mentions)
Sc 1616 r, by Santa Cruz Biotechnology (1 mentions)
Irdye secondary antibody, by LI COR (1 mentions)
Trans blot sd semi dry transfer cell, by Bio-Rad (1 mentions)
On targetplus smartpool sirna, by Horizon Discovery (1 mentions)
α tubulin t5168, by Merck Group (1 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions)
Adar1 antibody clone 15, by Santa Cruz Biotechnology (1 mentions)
12 protocols using sc 73408
1
ADAR Protein Expression Analysis
2
Depletion of NonO and Adar1 with siRNAs
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NonO (L-048587-00-0005, 40 nM) (ON-TARGETplus smartpool siRNA, GE Dharmacon, USA) and Adar1 siRNA (L-048587-00-0005, 150 nM) (ON-TARGETplus smartpool siRNA, GE Dharmacon, USA) were used to deplete NonO and Adar1, respectively. The siRNAs were transfected to cells using Lipofectamine RNAiMAX reagent as per the manufacturer’s instructions (Invitrogen, USA) and incubated for 48 hrs. Knockdown was confirmed using NonO antibody (gift from Dr Yasuyki Kurihara, Yokohama National University, Yokohama, Japan)44 (link) and ADAR1 antibody (sc-73408; Santa Cruz Biotechnology, USA). Loading controls used were α-tubulin (T5168, SIGMA, USA) and B”-U2snRNP.
3
ADAR1 Protein Detection in Mouse Brain
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Before tissues were collected from the mice, whole body perfusion was performed with phosphate buffered saline (PBS) with heparin to remove blood from the organs. The tissues were immediately frozen in liquid nitrogen until analysis. For the brain sample collections, the cerebellum and olfactory bulb were removed prior to freezing. Homogenization was performed in RIPA buffer with the addition of protease inhibitor cocktail. ADAR1 protein in the brain tissues was detected using western blot as described previously [18 (link)]. In brief, 30 ug of protein extract was loaded to each lane and separated on 8% polyacrylamide gel with 0.1% SDS. ADAR1 was detected with ADAR1 antibody clone 15.8.6 (Santa Cruz sc-73408) at 1:1,000 dilution.
4
ADAR1 and ISG15 Protein Analysis
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Cells were lysed in RIPA buffer (50 mM Tris.HCl, pH7.4; 150 mM NaCl; 1% NP-40 (Sigma-Aldrich), 0.5% Deoxycholate, 0.1% SDS and Complete protease inhibitor (Roche)) at 4°C for 10 minutes. Protein lysates were then cleared by centrifugation at 13000 rpm for 10 minutes. Samples were mixed with NuPAGE SDS-PAGE sample loading buffer (ThermoFisher) containing 10% 2-mercaptoethanol. A primary antibody against ADAR1 was purchased from Santa Cruz (sc-73408). The antibody recognizing ISG15 was a gift from Klaus-Peter Knobeloch. HRP-coupled secondary antibodies were from GE Healthcare.
5
Western Blot Analysis of ADAR Proteins
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Transduced cells were lysed using M-PER lysis buffer with 5 mM EDTA and HALT protease inhibitor cocktail (all Thermo Scientific) and cleared by centrifugation at 14,000g at 4°C for 10 min. Protein concentrations were determined using Coomassie Plus Bradford reagent (Thermo Scientific) and equal amounts of protein were prepared using Laemmli buffer (Bio-Rad) with added DTT. After denaturation at 95°C for 5 min, proteins were separated on 4%–20% Mini-Protean TGX gels (Bio-Rad) and blotted onto PVDF membranes using the TransBlot Turbo Transfer system (Bio-Rad). Membranes were blocked with blocking buffer (5% milk, 5% FBS, 1% BSA, 1 M Glycine) at room temperature and probed with primary antibodies at 4°C overnight. Primary antibodies were α-ADAR1 (sc-73408, Santa Cruz Biotechnology; 1:500), α-ADAR2 (sc-73409, Santa Cruz Biotechnology; 1:1000), and α-GAPDH (MAB374, Millipore; 1:25,000). Membranes were probed with α-mouse HRP secondary antibody (P0447, DAKO, Glostrup, Denmark) for 1 h at room temperature, and chemiluminescence was detected using Immobilon Forte Western HRP substrate (Millipore) on an Amersham Imager 600 (GE Healthcare).
6
Western Blot Analysis of RPTE Cells
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Total protein of RPTECs was prepared using CelLytic MT (Sigma-Aldrich) according to the manufacturer’s instructions. Denatured samples containing 5 μg of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride membranes. The membranes were incubated with primary antibodies against ADAR1 (1:1000, sc-73408; Santa Cruz Biotechnology), MRP4 (1:3000, ab15602; abcam), TBP (1:1000, ab51841; abcam), and β-ACTIN (1:10,000, sc-1616; Santa Cruz Biotechnology). Specific antigen–antibody complexes were visualized using horseradish peroxidase–conjugated anti-mouse antibodies (1:10,000, sc-2005; Santa Cruz Biotechnology) against ADAR1 and TBP, or anti-rat antibodies (1:10,000, ab97057; abcam) against MRP4 and ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation). Visualized images were scanned using ImageQuant LAS4010 (FUJIFILM).
7
Immunofluorescence Profiling of EMT Markers
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Tissue sections were incubated with primary antibodies labeled with fluorescent substance against ADAR1 (1:50, sc-73408, SANTA CRUZ BIOTECHNOLOGY, USA ), CALR (1:100; 27298-1-AP; Proteintech, China), β-catenin (1:100; 51067-2-AP; Proteintech, China), E-cadherin (1:150; ab231303; Abcam, USA) and Vimentin (1:1000; 10366-1-AP; Proteintech, China) overnight at 4 °C, followed by incubation with secondary antibodies Alexa Fluor® 488 Conjugate (1:100; #ZF-0512; ZSGB-BIO) as well as Alexa Fluor® 594 Conjugate (1:100; #ZF-0513; ZSGB-BIO) at room temperature lasting 2 h. The nuclear was counterstained by DAPI (ZLI-9557; ZSGB-BIO, China) for 5 min. The images were captured under a fluorescence microscope (BX61, Olympus, Japan).
8
Immunofluorescence Analysis of CD, ADAR1 in Tissue
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Immunofluorescence was performed on archival frozen sections of 3 patients with active CD, 3 with inactive CD and 3 controls. Samples were embedded in a cryostat mounting medium [Neg–50 Frozen Section Medium, Thermo Scientific], snap frozen and stored at −80°C. Sections 6 µm thick were mounted onto superfrost plus glass slides [Thermo Scientific] and fixed in 4% paraformaldehyde [PFA] for 10 min at 4°C. Slides were washed three times with PBS and treated with 0.1% Triton X-100 for 20 min at room temperature [RT]. Blocking was performed with a 10% normal goat serum PBS solution for 1 h at RT. Slides were then incubated overnight at 4°C with mouse anti-human CD3 (1:100, M3070 from Spring Bioscence); or hamster anti-human CD11c (1:75, MA11C5 from Invitrogen); and rabbit anti-human/mouse ADAR1 (1:100, sc-73408 from Santa Cruz Biotechnology). After washing three times with PBS, slides were incubated for 1 h at RT with specific secondary antibodies AF 488 goat anti-mouse (A11017), AF 568 goat anti-rabbit (A11011), AF 568 goat anti-hamster (A21112) all 1:2000 from Invitrogen. Coverslips were mounted on glass slides using ProLong Gold antifade reagent with DAPI [Invitrogen] to counterstain the DNA. Images were acquired on a Leica DMI 4000 B fluorescence microscope [Leica].
9
Immunohistochemistry Analysis of Cellular Markers
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Paraffin sections were incubated with primary antibodies against ADAR1(1:200, sc-73408, SANTA CRUZ BIOTECHNOLOGY, USA), CALR (1:100; 27298-1-AP; Proteintech, China), β-catenin (1:200; 51067-2-AP; Proteintech, China), E-cadherin (1:150; ab231303; Abcam, USA), MMP2 (1:150; ab92536; Abcam, USA), MMP9(1:100; 10375-2-AP; Proteintech, China), TGFβ (1:100; 21898-1-AP; Proteintech, China) and Vimentin (1:100; 10366-1-AP; Proteintech, China) at 4 ℃ overnight. Then, the sections were incubated with secondary antibodies conjugated with HRP (#SPN-9100; ZSGB-BIO, Beijing, China) at room temperature for 1 h. The sections were stained through a DAB kit (#ZLI-9018; ZSGB-BIO, Beijing, China). The immunohistochemical scores were achieved by two experienced pathologists in a double-blinded manner, as previously described 17 (link).
10
Western Blot Analysis of RPTE and Monkey Kidney
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Total protein of RPTECs and kidney of monkey was prepared using CelLytic MT (Sigma-Aldrich) according to the manufacturer’s instructions. Denatured samples containing 10 μg of protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride membranes. The membranes were incubated with primary antibodies against ADAR1 (1:1000, sc-73408; Santa Cruz Biotechnology), P-gp (1:1000, C494, Thermo Fisher Scientific), MRP2 (1:1500, sc-5770; Santa Cruz Biotechnology), TBP (1:1000, ab51841; abcam), and β-ACTIN (1:10,000, sc-1616; Santa Cruz Biotechnology). Specific antigen–antibody complexes were visualized using horseradish-peroxidase-conjugated anti-mouse antibodies (1:10,000, sc-2005; Santa Cruz Biotechnology) against ADAR1, P-gp and TBP, or anti-goat antibodies (1:10,000, sc-2020; Santa Cruz Biotechnology) against MRP2 and ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation). Visualized images were scanned using an ImageQuant LAS3000 (FUJIFILM).
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