Estimation of the Epo and EpoR protein expression was performed by western blot analysis. Total lysis of the cells and spinal cords was conducted using the Radio-Immunoprecipitation Assay buffer (Thermo Fisher Scientific, Inc.), and protein concentrations were determined using the bicinchoninic acid protein assay (Thermo Fisher Scientific, Inc.). Equal amounts (20 µg) of the proteins were separated by SDS-PAGE gel, which were subsequently electroblotted onto nitrocellulose membranes. The membranes were blocked with 5% skim milk and were probed overnight at 4°C with the primary antibodies anti-Epo (1:800; Santa Cruz Biotechnology, Inc.) and anti-EpoR (1:800; Santa Cruz Biotechnology, Inc.). The membrane was then incubated with secondary horseradish peroxidase-conjugated antibody (1:2,000; Santa Cruz Biotechnology, Inc.) at room temperature for 2 h. The membranes were then prepared using an electrochemiluminescence western blotting kit (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. The optical density value of each band was quantified using ImageJ and was normalized to the corresponding β-actin level. Values were expressed as the fold change relative to the control value.
Anti epo
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-EPO is a laboratory product used to detect and quantify the presence of erythropoietin (EPO) in biological samples. It is a crucial tool for researchers studying the regulation and function of EPO, a hormone that stimulates the production of red blood cells.
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Lab products found in correlation
Anti epor, by Santa Cruz Biotechnology (3 mentions)
Bicinchoninic acid protein assay, by Thermo Fisher Scientific (1 mentions)
Radioimmunoprecipitation assay buffer, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Nb100 132, by Novus Biologicals (1 mentions)
Mab1536, by R&D Systems (1 mentions)
Anti xpress, by Thermo Fisher Scientific (1 mentions)
Anti lsd1, by Santa Cruz Biotechnology (1 mentions)
Anti ki67, by Cell Signaling Technology (1 mentions)
Anti hif 2α, by Novus Biologicals (1 mentions)
Anti hif 1α, by Novus Biologicals (1 mentions)
Anti flag, by Merck Group (1 mentions)
Anti caspase 3, by Cell Signaling Technology (1 mentions)
Anti ha mms 101r, by Fortrea (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Nb100 122, by Novus Biologicals (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Tnb blocking buffer, by PerkinElmer (1 mentions)
Anti jak2, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
9 protocols using anti epo
1
Western Blot Analysis of Epo and EpoR
2
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).
3
Quantitative Western Blot Analysis
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Twenty-four micrograms of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and subsequently blotted onto a polyvinylidene fluoride (PVDF) membrane. The transferred membranes were blocked in 4% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) and then incubated with primary antibodies overnight at 4°C. Anti-EPO and Anti-EPOR were obtained from Santa Cruz Biotechnology, and the anti-neuron-specific enolase (anti-NSE) antibody was purchased from Abcam (Cambridge, MA, USA). Following incubation with the primary antibody, the membranes were rinsed and incubated with the HRP-conjugated anti-rabbit secondary antibody and developed using a standard enhanced chemiluminescent (ECL) system, and the signal was captured by an Amersham Imager 600 (GE Healthcare, Madison, WI, USA). All of the procedures were performed in triplicate.
4
Radiation-Induced Spleen Protein Analysis
5
EPO and SST Immunohistochemistry Protocol
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6
Immunostaining of EPO, EpoR, JAK2, and STAT3
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For EPO, EpoR, JAK2, and STAT3 immunostaining, slices (n = 6 for each group) were pretreated with Tris-Na-Blocking (TNB) blocking buffer (PerkinElmer, USA) containing 0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl, and 0.5% blocking reagent before incubation with a primary antibody. Free-floating sections were immunolabeled with the following rabbit polyclonal antibodies from Santa Cruz Biotechnology (USA) overnight at 4°C: anti-EPO (Epo; 1 : 50); anti-EPOR (1 : 50); anti-JAK2 (1 : 100); and anti-STAT3 (1 : 100). Then, the PV-6001 Polink-1 HRP DAB Detection System, one-step polymer detection system, for mouse, rabbit, and rat antibodies was used at 37°C for 1 h according to manufacturer's instructions (GBI, Inc., USA). The slices were visualized by incubating with 0.5% diaminobenzidine (DAB). Finally, the number of immune positive cells in the penumbra area was calculated under a light microscope.
7
Quantitative Analysis of Erythropoietin Expression
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BMDMs were conditioned overnight with apoptotic cells or DMEM culture media. The next day, BMDMs were washed three times with PBS and were incubated with Brefeldin A (Biolegend) for 4 h. Afterwards, cells were lysed with RIPA buffer (NaCl 150 mM, Tris 50 mM, NP40 1%, sodium deoxycholate 0.5%, SDS 0.1%). Protein concentration was determined with DC protein assay (Bio-Rad) and 10 µg of total protein was added per wells for SDS-PAGE. Transfer was performed with transblot turbo (Bio-Rad) using manufacturer standard semi-dry transfer protocol onto PVDF membrane (Millipore, Oakville, Canada). Membrane was blocked overnight with 3% BSA in TBS-T and probed with 1:1000 anti-EPO (Santa Cruz, Dallas, TX, USA, cat#SC-5290) overnight at 4 °C. After revelation, the membrane was stripped, blocked, and probed with 1:50,000 anti-beta actin (Sigma Aldrich) for 1 h at room temperature. Femto super signal ECL (Pierce, Waltham, MA, USA) was used as substrate and revelation was captured using the Chemidoc MP imaging system (Bio-Rad). Densitometry analysis were performed using image lab software (Bio-Rad) and EPO expression was normalized to beta-actin.
8
Immunoprecipitation of Cellular Proteins
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Cells lysates in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and protease inhibitors cocktail) were centrifuged at 15,000 rpm for 30 minutes at 4°C, and 1 mg protein of cleared lysates were used for each immunoprecipitation. The lysates were incubated overnight at 4°C with primary antibodies and then 30 μl of protein A/G PLUS-agarose beads (Santa Cruz) were added and incubation continued. Beads were washed with cold lysis buffer and boiled in SDS-PAGE sample buffer before elctrophoresis. Primary antibodies used were anti-ZFP91 [5 (link)], anti-HIF-1α (BD Biosciences; 610959 and Novus Biologicals; NB100-105), anti-VEGF (Santa Cruz; sc-152), anti-EPO (Santa Cruz; sc-80995), anti-CD31 (Santa Cruz; sc-1506), anti-p65 (Santa Cruz; sc-109), anti-Topo-I (Santa Cruz; sc-5342), anti-α-tubulin (Sigma; T5168).
9
Quantifying Extracellular DNA in BALF
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Extracellular DNA levels in BALF supernatant was determined by the Quant-iT dsDNA HS kit (Invitrogen). To visualize the EETs formation by immunofluorescence microscopy, BALF eosinophils (2 × 10 5 /ml) were plated in eight-chamber culture slides and incubated at 37°C with 5% carbon dioxide for 1 hr. Afterward, BALF eosinophils were incubated with 4% paraformaldehyde (PFA) for 45 min. Next BALF eosinophils were incubated for 45 min with primary antibodies, anti-EPO and anti-histone H2B (1:250; Santa Cruz Biotechnology). After BSA 5%; Cell Signaling Technology), or anti-β-Actin (1:1000 in BSA 5%; Cell Signaling Technology) overnight. Finally, it was incubated with secondary antibody, horseradish peroxidase anti-rabbit (1:1000 in BSA 5%; Cell signaling). The blot was developed using a Chemiluminescent photo finder (Kodak/Carestream). The bands were normalized by β-actin using Image J (Rueden et al., 2017) (link).
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