All the necessary experimental procedures were approved and undertaken in accordance with guidelines of Stanford University, Forsyth Institute, Gauhati University and Kavi Krishna laboratory institutional animal ethics committee. Seven such transgenic mice were selected for the study and genotype confirmed (Supplementary table 1 ). The generation and genotyping of Eu-tTA/tetO-MYC system transgenic lines for conditional MYC-driven lymphoma has been used as described (34 (link)). The thymus obtained from moribund animals were dissociated to flow cytometry or immunomagnetic sort Sca-1+ cells (36 (link)) and these cells were expanded in serum free media containing IL-7 and SCF, and then subjected to phenotypic analysis. Multi-color flow cytometry for HIF-2α (#NB100–132, Novus Biologicals, CO) and Nanog (#ab184609, Abcam, MA) was done as described(33 (link)).
Nb100 132
Manufactured by Novus Biologicals
Sourced in United States
NB100-132 is a laboratory product offered by Novus Biologicals. It is a primary antibody that can be used for research purposes. The core function of this product is to serve as a tool for scientific investigation, though its specific intended use should not be extrapolated beyond this factual description.
Automatically generated - may contain errors
Lab products found in correlation
Nb100 105, by Novus Biologicals (4 mentions)
Nb100 134, by Novus Biologicals (2 mentions)
Ab2185, by Abcam (2 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (2 mentions)
Sc 8076, by Santa Cruz Biotechnology (2 mentions)
Alexafluor 488 anti rabbit immunoglobulin, by Thermo Fisher Scientific (2 mentions)
Krt10, by Fortrea (2 mentions)
Ab34771, by Abcam (1 mentions)
Ab8158, by Abcam (1 mentions)
Ab290, by Abcam (1 mentions)
Nb 600 749, by Novus Biologicals (1 mentions)
Af3159, by R&D Systems (1 mentions)
Af4488, by R&D Systems (1 mentions)
Sc 25600, by Santa Cruz Biotechnology (1 mentions)
Ab234, by Evrogen (1 mentions)
Tcs sp5, by Leica (1 mentions)
Deferoxamine, by Merck Group (1 mentions)
Nb100 449, by Novus Biologicals (1 mentions)
Anti actin sc 1616, by Santa Cruz Biotechnology (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
15 protocols using nb100 132
1
Conditional MYC-driven Lymphoma Analysis
2
Histological and Molecular Characterization
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At the experimental end-point animals were euthanized and tissues were fresh frozen in liquid nitrogen or fixed in 4% paraformaldehyde in PBS overnight. To review histology, slides were stained by haematoxylin and eosin (H&E). Immunohistochemistry (IHC) and immunofluorescence (IF) were performed using standard protocols. Specificity of immunostaining was assessed by incubation in the absence of primary antibody. We used the following primary antibodies: RFP for Strawberry detection (ab34771, 1:400; Abcam), Aquaporin 1 (NB-600–749, 1:500; Novus Biologicals), THP (AF5175, 1:100; R&D), Aquaporin 2 (ab105171, 1:1000; Abcam), Nephrin (AF3159, 1:100; R&D), CD34 (ab8158, 1:50; Abcam), CD73 (AF4488, 1:100; R&D), GFP (ab290, 1:1000; Abcam), HIF2a (NB100-132, 1:150; Novus Biologicals), CA9 (sc-25600, 1:200; Santa Cruz), turbo-RFP (AB234, 1:500; Evrogen) and pS6 (2211, 1:200; Cell Signalling). Secondary antibodies used were conjugated to HRP (IHC) or Alexa-fluor® fluorochromes (IF). Fluorescent images were obtained by confocal laser-scanning microscopy (Leica TCS SP5).
3
Isolation and Analysis of Nuclear Proteins from Murine Retinas
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Nuclear protein extract were prepared as described previously [31] (link). Briefly, neonatal mice were euthanized by decapitation, and retinas were isolated immediately in pre-chilled PBS, which was frequently replaced with additional aliquots of PBS precooled on ice. Nuclear protein extracts were prepared by homogenizing retinas in ice cold nuclear extraction buffer (10 mM HEPES-KOH (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 0.2 mM deferoxamine (Sigma), 0.1% NP-40, and 1× complete protease inhibitor cocktail (Roche)). Nuclei were collected by centrifugation, and resuspended in ice cold re-suspension buffer containing 20 mM HEPES-KOH (pH 7.9), 420 mM NaCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol, 0.2 mM deferoxamine, 1× protease inhibitor cocktail, 0.2 mM phenylmethylsulfonyl fluoride, and 25% glycerol. The following antibodies were used for Western blotting: anti-HIF-1α (NB100-449, Novus Biologicals), anti-HIF-2α (NB100-132, Novus Biologicals), and anti-ß-actin (sc-1616; Santa Cruz Biotechnology).
4
Antibody Characterization for Hypoxia Signaling
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The following commercially available antibodies were used: anti-HIF-1α (NB100–132, Novus; 10006421, 1:1,000 dilution for IB analysis, Cayman; 1:1,000 dilution for IB analysis, 1:200 for IF analysis; MAB 1536, R&D Systems, 1:1,000 dilution for IB analysis); anti-HIF-2α (NB100–122, Novus, 1:1,000 dilution for IB anlysis); anti-Xpress (R910-25, Invitrogen, 1:5,000 dilution for IB analysis); anti-FLAG (F3165, Sigma, 1:10,000 dilution for IB analysis); anti-methyl-Lys (ab23366, Abcam); anti-CD31 (clone 2H8, MAB1398Z, Millipore, 1:200 dilution for immunohistochemical (IHC) analysis); anti-HA (MMS-101R, Covance, 1:5,000 dilution for IB analysis); anti-VEGF (AF493NA, R&D System, 1:200 dilution for IHC analysis); anti-EPO (sc-7956, 1:1,000 for IB analysis), anti-Brn3b (sc-6026, 1:200 dilution for IHC analysis) from Santa Cruz; anti-LSD1 (#2139, 1:1,000 dilution for IB analysis), anti-hydroxyl-HIF-1α (#3434, 1:5,000 dilution for IB analysis), anti-Caspase3 (#9661, 1:200 dilution for IHC analysis), anti-Ki-67 (#9027, 1:100 dilution for IHC analysis) and anti-SET7/9 antibodies (#2813, 1:1,000 dilution for IB analysis) from Cell Signalling. anti-HIF-1α-K32 methyl antibodies were generated by Abfrontier (South Korea, 1:5,000 dilution for IB analysis).
5
HIF-1α and HIF-2α Interaction with ALK5
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On the day preceding transfection, ACHN (5x105) and A498 (5x105) cells were seeded onto two well chamber slide. ACHN cells were transfected with ALK5-HA or co-transfected with siVHL and ALK5-HA. A498 cells were transfected with ALK5-HA or co-transfected with ALK5-HA and VHL vectors. Cells transfected with ALK5-HA were treated with TGF-β (10 ng/ml) for 6h. In both cell lines, cells were transfected with ALK5-HA, but not incubated with one of the primary antibodies during PLA and thus served as a negative control for PLA. PLA was performed using Duolink® proximity ligation assay (PLA®, Sigma-Aldrich) following the manufacturer’s instructions. In brief, cells were washed with PBS and then fixed with 4% formaldehyde. Triton-X was used to permeabilize the cells, and 5% BSA was used for blocking. The following primary antibodies were used: HIF-1α (NB100-134, Novus Biologicals), HIF-2α (NB100-132, Novus Biologicals), HA (CST #3724, Cell Signaling Technology) and HA (CST #2367, Cell Signaling Technology). Rabbit plus and mouse minus probes were used against the primary antibodies. PLA signal was obtained after scanning the slides using Zeiss 710 Meta microscope and ZEN-2010 software (Jena, Germany). Quantification of the graphic representation was performed using the Duolink image tool (Sigma-Aldrich).
6
Immunostaining of Paraffin Sections
7
IP of ALK5 and HIF-α Regulation
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For immunoprecipitation (IP), ACHN cells were transfected with ALK5-HA or co-transfected with ALK5-HA and siVHL, followed by starvation for 12h and stimulation with TGF-β for 6h. A498 cells were transfected with ALK5-HA or co-transfected with ALK5-HA and VHL, followed by starvation for 12h and stimulation with TGF-β for 6h.
For IP, total cell lysate was prepared from the collected cells and immunoprecipitated using the indicated antibody. Immunoblotting was performed and then probed with either HA (CST #2367) or HIF-1α (NB100-134, Novus Biologicals) or HIF-2α (NB100-132, Novus Biologicals) or ALK5 (V22) antibody. The primary antibodies were detected by Quick Western Kit–IRDye® 680RD (#926–68100, Licor Biosciences) following manufacturer’s instructions.
For IP, total cell lysate was prepared from the collected cells and immunoprecipitated using the indicated antibody. Immunoblotting was performed and then probed with either HA (CST #2367) or HIF-1α (NB100-134, Novus Biologicals) or HIF-2α (NB100-132, Novus Biologicals) or ALK5 (V22) antibody. The primary antibodies were detected by Quick Western Kit–IRDye® 680RD (#926–68100, Licor Biosciences) following manufacturer’s instructions.
8
Protein Extraction and Western Blotting
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Cells were harvested with RIPA lysis buffer containing 0.5% NP-40, 150 mM NaCl, 1 mM EDTA (pH 8.0), 50 mM Tris-HCl (pH 8.0), and protease inhibitors. Protein extracts were quantified and separated by electrophoresis on a 15% SDS–PAGE gel. Primary antibodies including HIF1α (1:1000, NB100–105, Novus, Littleton, CO, USA), HIF2α (1:500, NB100–132, Novus), GAPDH (1:10,000, SC32233, Santa Cruz Biotechnology, Santa Cruz, CA, USA), α-tubulin (1:10,000, bs1699, Bioworld, Dublin, OH, USA), GSK3β (1:1000, Cell Signaling Technology, 9332, Danvers, MA, USA), p-GSK3β (1:1000, Cell Signaling Technology, 9336), ERK1/2 (1:1000, bs1112, Bioworld), and p-ERK1/2 (1:1000, bs4621, Bioworld) were used. The secondary antibodies used were goat anti-mouse (1:10,000, 31,439, Pierce, Rockford, IL, USA) and goat anti-rabbit (1:10,000, Sigma-Aldrich).
9
Protein Isolation and Western Blot Analysis
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Tissues were lysed in RIPA buffer (20 mM Tris pH 7.5, 150 mM sodium chloride, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA, 0.1% SDS) containing complete mini protease inhibitors (Roche) and phosphatase inhibitors. Western blots were performed using 20~50 μg of lysate protein, and membranes were incubated with antibodies against HIF-1α (1:500; NB 100-105, Novus, CO, USA) or HIF-2α (1:1000; NB 100-132, Novus, CO, USA).
10
Immunofluorescence Staining of Cellular Markers
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For immunofluorescence staining, cells were fixed in 4% paraformaldehyde for 10 min, incubated in blocking solution (5% goat serum and 0.5% Triton X-100 in PBS) for 30 min and then incubated with unconjugated anti-α-SMA antibody (A5228; Sigma-Aldrich, St Louis, MO, USA), anti-HIF-2α antibody (ab109616; Abcam or NB100-132; Novus) or anti-CD31 antibody (ab28364; Abcam). Cells were then incubated with Alexa-Fluor 488 (green) conjugated anti-mouse IgG2a antibody (Invitrogen, Carlsbad, CA, USA), Alexa-Fluor 568 (red) conjugated anti-mouse IgG antibody (ab1500113; Abcam), or Alexa-Fluor 568 (red) conjugated anti-rabbit IgG antibody (ab175471; Abcam) and with Hoechst 33342 (Sigma-Aldrich) for 60 min. Cells were imaged and images captured with an IX83 microscope (Olympus, Tokyo, Japan) and the ratios of α-SMA- or HIF-2α-positive cells to total cell numbers estimated from Hoechst 33342 nuclear staining were calculated using Image-J (National Institutes of Health, Bethesda, MD, USA).
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