For determination of phospho-p38/-p44/42 (Cell Signaling, Danvers), cells were prepared with western blot lysis buffer (125 mM Tris-HCl pH 7.8, 20% glycerol, 4% SDS (sodium dodecyl sulfate), 0.1 M dithiothreitol, bromphenol blue, Sigma Aldrich, St. Louis) at the indicated time points. Samples were analyzed by SDS-Polyacrylamide gel electrophoresis (SDS-PAGE). Afterwards, proteins were transferred to nitrocellulose membranes (Whatman Inc., Florham Park, NJ). Membranes were blocked with TBS (0.1% Tween)/5% fat-free skimmed milk and incubated with the respective antibodies. For detection, a horseradish peroxidase-linked anti-mouse IgG antibody (Cell Signaling) and enhanced chemiluminescence substrate (Thermo Fisher Scientific, Rockford, IL) were used. Images were acquired by Fusion FX7 (Vilber Lourmat, Eberhardzell, Germany) and the density of each band was measured by Bio-1D software (Vilber Lourmat). Equal loading and blotting efficiency were verified by an anti-β-actin antibody and pre-stain marker (Cell Signaling). All data are the average from seven independent experiments and the error bars represent the standard error of the mean (s.e.m.).
Anti mouse igg horseradish peroxidase linked antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-mouse IgG horseradish peroxidase-linked antibody is a secondary antibody used in various immunological techniques. It is designed to detect and bind to mouse primary antibodies, allowing for their visualization through a horseradish peroxidase (HRP) enzyme-mediated colorimetric or chemiluminescent reaction.
Automatically generated - may contain errors
Lab products found in correlation
Bromphenol blue, by Merck Group (2 mentions)
Bio1d software, by Vilber (2 mentions)
Fusion fx7, by Vilber (2 mentions)
Anti rabbit igg horseradish peroxidase linked antibody, by Cell Signaling Technology (2 mentions)
Enhanced chemiluminescence substrate, by Thermo Fisher Scientific (1 mentions)
Anti hif 1α antibody, by Cell Signaling Technology (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Anti flag m2 antibody, by Merck Group (1 mentions)
Ecl chemiluminescence system, by GE Healthcare (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin, by Nacalai Tesque (1 mentions)
Streptomycin, by Nacalai Tesque (1 mentions)
Insulin transferrin selenium, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium f 12, by Fujifilm (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Anti rabbit immunoglobulin g igg horseradish peroxidase linked antibody, by Cell Signaling Technology (1 mentions)
Amersham ecl select, by Cytiva (1 mentions)
Amersham imagequant 800, by Cytiva (1 mentions)
11 protocols using anti mouse igg horseradish peroxidase linked antibody
1
Quantitative Analysis of Phospho-p38/-p44/42 Proteins
2
Western Blot Analysis of HIF-1α and pp70
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The cells were lysed in lysis buffer (125 mM Tris-HCl pH = 7.8, 20% glycerol, 4% SDS, 0.1 M dithiothreitol, bromphenol blue (Sigma - Aldrich). Protein separation was performed on 4% polyacrylamide gel and electro blotted on nitrocellulose membrane (Whatman Inc., Florham Park, NJ.) TBS (0.1% Tween)/5% fat-free milk was used for membrane blockade. Membranes were incubated with anti-HIF-1α-antibody or anti-pp70-antibody at 4 °C overnight, followed by horseradish peroxidase- linked anti-mouse IgG antibody (both from Cell Signaling Technologies, Danvers, MA), and enhanced chemiluminescence substrate (Thermo Fisher Scientific), before analysis by an automated image acquisition system (Fusion FX7, Vilber Lourmat, Eberhardzell, Germany). Protein amounts were quantified using Bio1D software (Vilber Lourmat). Anti-β-actin (Cell Signaling Technologies) served as loading and blotting control in all experiments. In case additional samples are displayed on the initial blots that are not relevant to this study, the images were cropped without additional modifications before analysis. Full-length blots are included in Figure S5 .
3
Monitoring eIF2α Phosphorylation and Ded1 Turnover
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To monitor levels of phosphorylated eIF2α (Sui2), anti-phospho-EIF2S1 (Ser52) polyclonal antibody (#44–728G; Thermo Fisher Scientific) and horse radish peroxidase-linked anti-rabbit IgG antibody (406401; BioLegend) were used. The levels of FLAG-tagged Ded1, Sui2, and Gcn4 were analyzed via western blotting using an anti-FLAG M2 antibody (F1804; Sigma-Aldrich) and horse radish peroxidase-linked anti-mouse IgG antibody (7076S; Cell Signaling Technology). Ded1 turnover was examined using a cycloheximide-chase assay (76 (link)). The aggregate sedimentation assay was performed to monitor the levels of aggregated Ded1. Collected cells were disrupted with glass beads in lysis buffer (50 mM potassium phosphate buffer [pH 6.8], 1 mM ethylenediaminetetraacetic acid, 5% glycerol, and 1× Protease inhibitor [Roth]) to prepare the cell-free extract. Aggregated Ded1 protein was collected via centrifugation of the cell-free extract at 16,000g for 20 min at 4 °C and then resuspended in urea buffer (50 mM Tris-HCl [pH 7.5], 6.0 M urea, and 5% sodium dodecyl sulphate). The bands on the western blots were quantified using the ImageJ software (http://imagej.nih.gov/ij/ ) and normalized to total protein levels or EzStainAQua MEM (EzSAQ) staining (WSE-7160; ATTO Corporation).
4
Quantification of RGMa Protein in Primate Tissues
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Following sedation with ketamine hydrochloride (10 mg/kg, i.m.) and xylazine hydrochloride (1 mg/kg, i.m.), an intact monkey was anesthetized deeply with sodium pentobarbital (50 mg/kg, i.v.) and perfused transcardially with 0.1 M phosphate-buffered saline (PBS; pH 7.4). The MI (digit region) and the spinal cord (C7 to Th1 segments) were harvested and homogenized in a lysis buffer, comprising 50 mM Tris-HCl (pH 7.8) with 150 mM NaCl, 1 mM EDTA, 2 mM Na3VO4, 1% NP-40, and protease inhibitor cocktail. After centrifugation at 13,000 g for 20 min at 4˚C, it was lysed by using equal amounts of 2 x sample buffer, comprising 250 mM Tris-HCl, 4% sodium dodecyl sulphate, 20% glycerol, 0.02% bromophenol blue, and 5% β-mercaptoethanol, and then proteins were boiled for 5 min at 95˚C. The mixed proteins were separated on sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to a polyvinylidence fluoride membrane (Millipore). The membrane was blocked with 5% skim milk in 0.01 M PBS containing 0.05% Tween-20 and then incubated with anti-RGMa antibody (IBL). After several washes, the membrane was incubated with horseradish peroxidase-linked anti-mouse IgG antibody (1:5,000; Cell Signaling Technology). The detection was carried out with an ECL chemiluminescence system (GE Healthcare). As control samples, human-and mouse-recombinant RGMa proteins (R & D systems) were used.
5
Antibody Sourcing for Cellular Studies
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Antibodies were obtained from the following suppliers: anti-Mas receptor (AAR-013) (Alomone Laboratories, Ltd., Jerusalem, Israel); anti-endothelial nitric oxide synthase (eNOS) (BD Biosciences, Mississauga, ON, Canada); anti-aryl hydrocarbon receptor (AhR) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-α-tubulin (Calbiochem, La Jolla, CA, USA); anti-signal transducer and activator of transcription 3 (Stat3), anti-phosphorylated eNOS (Ser1177) (peNOS), anti-transforming growth factor-β1 (TGF-β1), anti-rabbit IgG horseradish peroxidase-linked antibody and anti-mouse IgG horseradish peroxidase-linked antibody (Cell Signaling Technology, Beverly, MA, USA). IS was obtained from Alfa Aesar (Morecambe, Lancs., UK). Ang-(1–7) was purchased from Bachem California (Torrance, CA, USA). N-acetylcysteine (NAC), an antioxidant, was from Calbiochem (La Jolla, CA, USA). Dulbecco’s modified Eagle’s medium/F12 was purchased from Wako (Osaka, Japan). Trypsin-EDTA, fetal bovine serum, and insulin transferrin-selenium were purchased from Gibco (Grand Island, NY, USA). Penicillin and streptomycin were purchased from Nacalai Tesque Inc. (Kyoto, Japan).
6
Platelet Protein Expression Analysis
7
Detecting Nuc1-HA Protein in Sporidia
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Hemagglutinin (HA)-tagged Nuc1 protein was detected as previously described (Tanaka et al., 2014 (link); Tanaka et al., 2019 (link)). Sporidia cells were incubated until OD600 reaches to 1.0 in 4 ml YEPSL medium. After centrifugation, the cell pellet was used for the extraction of total proteins. Culture supernatant was filtered with 0.2 μm filter to remove residual sporidia cells. Secreted Nuc1-HA protein was harvested by TCA precipitation from culture supernatant as previously described (Tanaka et al., 2014 (link)). Proteins were separated by 12% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis. A rabbit polyclonal anti-HA antibody (H6908; Sigma-Aldrich, St. Louis, MO, U.S.A.) or a mouse monoclonal anti-tubulin antibody (T9026; Sigma-Aldrich) were used as the primary antibody at 1: 10000 dilution to detect Nuc1-HA or tubulin protein, respectively. Anti-rabbit immunoglobulin G (IgG) horseradish peroxidase-linked antibody (Cell Signaling Technology, Danvers, MA, U.S.A.) or anti-mouse IgG horseradish peroxidase-linked antibody (Cell Signaling Technology) were used as a secondary antibody at 1: 10000 dilution. To detect signals, Amersham ECL select (Cytiva) was used as substrate for horseradish peroxidase and the signal was detected by Amersham ImageQuant 800 (Cytiva).
8
Immunoblotting of Transfected HEK293 Cells
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HEK293 cells were seeded on 6-well culture plates (Techno Plastic Products, Trasadingen, Switzerland) and transfected with 1 μg of vector constructs. Forty-eight hours after transfection, the cells were washed twice with PBS and harvested in RIPA buffer containing 1× Protease Inhibitor Cocktail Set (Fujifilm Wako). Cell lysates were mixed with an SDS sample buffer. Equal amounts (0.5 μg) of protein samples were loaded in the respective lanes and electrophoresed on gradient polyacrylamide gels (ATTO Corporation, Tokyo, Japan). Electrophoresed proteins were transferred to polyvinylidene fluoride membranes and reacted with anti-DYKDDDDK (Clone # 2H8; dilution rate, 1:1000; TransGenic, Fukuoka, Japan) or anti-ERβ (PPZ0506; 1:2000; Perseus Proteomics, Tokyo, Japan) monoclonal antibodies in 0.3% skim milk/0.1% Tween-20/Tris-buffered saline (TBS; 25 mM Tris-HCl (pH 7.4), 0.15 NaCl) for 16 h. After washing three times in 0.1% Tween-20/TBS, the membranes were incubated with anti-mouse IgG, horseradish peroxidase-linked antibody (Lot # 31; 1:2500; Cell Signaling Technology, Danvers, MA, USA) in 0.3% skim milk/0.1% Tween-20/TBS for 1 h. Immunoluminescent signals were detected with ImmunoStar Zeta substrates (Fujifilm Wako) using an ImageQuant LAS 4000 System (GE Healthcare, Chicago, IL, USA).
9
Protein Extraction and Western Blotting
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Proteins were extracted from mixed stage cultures. Concentration was determined by Neuhoff's Dot-Blot assay24 (link). Proteins were equally loaded and separated in polyacrylamid gels. Proteins were transferred to polyvinylidene difluoride transfer membrane and incubated overnight with primary antibodies (Supplementary Table 3 ), and were then incubated for an hour in secondary antibodies (Anti-rabbit IgG, horseradish peroxidase-linked antibody, Cell Signaling Technology, Cat. #7074S and Anti-mouse IgG, horseradish peroxidase-linked antibody, Cell Signaling Technology, Cat. #7076S). For dilution of primary antibodies see Supplementary Table 3 . The secondary antibody was diluted 1:1,000. The detection was done by Bio-Rad Clarity western enhanced chemiluminescent (ECL) substrate using Peqlab FUSION Xpress multi-imaging system. We provide an uncropped scan of the most important blot as Supplementary Fig. 5 .
10
Time-course Analysis of Viral Protein Expression
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Overnight cultures were diluted 1:100 in fresh LB medium and incubated at 37 °C with shaking (250 rpm) until an OD600 of 0.4 was reached. Cultures were infected with DMS36HisTT at an MOI of two. At timepoints 0, 30, and 60-min post-infection, 5 mL of cells were collected by centrifugation and flash frozen at −80 °C. The bacterial pellets were resuspended in 100 μL ice-cold sonication lysis buffer and were sonicated at 30 kHz for 5 min (30 s on, 30 s off). Cleared cell lysates were mixed with 2X Laemmli SDS loading dye (Bio-Rad), boiled at 100 °C for 20 min and then separated by 15% SDS-PAGE. For Western blot analyses, proteins on SDS-PAGE gels were transferred to a 0.45 μm nitrocellulose membrane using Bio-Rad Trans-lot SD Semi-Dry Transfer Cell at 15 V for 1 h. Western blots were incubated with primary mouse anti-His6 monoclonal antibody (BioShop TAG001.100; diluted with TBS-T buffer containing Bovine serum albumin at 1:5,000) at 4 °C overnight, and then with secondary anti-Mouse IgG Horseradish peroxidase-linked antibody (Cell Signalling Technology; diluted with TBS-T buffer containing Bovine serum albumin at 1:10,000). Western blots were developed using Bio-Rad ClarityTM Western ECL substrate (Cat. # 170-5060) and imaged with the Bio-Rad ChemiDoc Imaging system.
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