Primary cells and brain tissue were homogenized in 10 volumes of RIPA buffer (Thermo Scientific, Waltham, MA, USA) containing protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA) and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Western blotting was performed as previously described. The membranes were immunoblotted with primary antibodies, followed by incubation with horseradish peroxidase-conjugated anti-rabbit and anti-mouse secondary antibodies (Jackson Laboratories). The antibodies used in this study are listed in Table 2 . Band densities were measured using ImageJ software (NIH, Bethesda, MD, USA) and normalized to the density of the actin band.
Horseradish peroxidase conjugated anti rabbit and anti mouse secondary antibodies
Manufactured by Jackson ImmunoResearch
Sourced in United States
Horseradish peroxidase-conjugated anti-rabbit and anti-mouse secondary antibodies are laboratory reagents used in various immunoassays and detection techniques. These antibodies bind to primary antibodies raised in rabbit or mouse, and the conjugated horseradish peroxidase enzyme can be used to visualize and quantify target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Merck Group (2 mentions)
Phosphatase inhibitor, by Merck Group (2 mentions)
Radio immunoprecipitation assay ripa buffer, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Matrigel, by BD (1 mentions)
Alexa fluor 488 and 594 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Texas red 70 kda dextran, by Thermo Fisher Scientific (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
C myc, by Abcam (1 mentions)
Lightcycler 480 probes master, by Roche (1 mentions)
Cell death detection elisaplus kit, by Roche (1 mentions)
Cleaved caspase 3 antibody, by Cell Signaling Technology (1 mentions)
α tubulin, by Cell Signaling Technology (1 mentions)
P jnk jnk, by Cell Signaling Technology (1 mentions)
Ecl western blotting detection reagent, by GE Healthcare (1 mentions)
Procaspase 3, by Cell Signaling Technology (1 mentions)
Nf κb oligonucleotide, by Promega (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
6 protocols using horseradish peroxidase conjugated anti rabbit and anti mouse secondary antibodies
1
Western Blotting of Brain Tissue Proteins
2
Western Blot Analysis of Protein Levels
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N2A cells and brain tissues were homogenized in 10 volumes of radioimmunoprecipitation assay (RIPA) buffer (Thermo Scientific, Waltham, MA, USA) containing protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA) and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA). A Western blot analysis was performed as previously described [64 (link)]. The membranes were immunoblotted with primary antibodies, followed by incubation with horseradish peroxidase-conjugated antirabbit and antimouse secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). The antibodies used in this study are listed in Supplementary Table S2 . The band densities were measured using ImageJ software (version 1.53, National Institutes of Health, Bethesda, MD, USA) and normalized to the density of actin.
3
Western Blot Analysis of Protein Expression
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Both the F3 and F3.ChAT cells, as well as brain tissues, were homogenized in 10 volumes of Radioimmunoprecipitation Assay (RIPA) buffer (Thermo Fisher Scientific, Waltham, MA, USA), supplemented with protease (Sigma-Aldrich) and phosphatase (Sigma-Aldrich) inhibitors. The Western blotting procedure was performed as previously described [12 (link)]. Membranes were subjected to immunoblotting with the appropriate primary antibodies, which was followed by incubation with horseradish peroxidase-conjugated anti-rabbit and anti-mouse secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). The specific antibodies utilized in this study are detailed in Supplementary Table S2 . Band densities were quantified using ImageJ software (NIH, Bethesda, MD, USA) and were normalized to the actin controls.
4
Monoclonal Antibody and Cell Culture Reagents
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Monoclonal hemagglutinin (HA) antibody, rat platelet-derived growth factor-BB, cell culture grade glutamine and penicillin-streptomycin were all from Sigma-Aldrich (St. Louis, MO). Monoclonal c-myc (9E10) antibody was from Roche Applied Science (Indianapolis, IN). FuGENE 6 transfection reagent was from Roche Diagnostics (Indianapolis, IN). AlexaFluor 488- and 594-conjugated secondary antibodies, AlexaFluor 594- conjugated phalloidin, and Texas-Red dextran (70 kDa) were from Invitrogen (Carlsbad, CA). Horseradish peroxidase-conjugated anti-rabbit and anti-mouse secondary antibodies were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Matrigel was from BD Biosciences.
5
Western Blot Analysis of ASF1A, c-Myc, and HES1
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Cells were collected, washed twice in phosphate-buffered saline (PBS), and then lysed for 30 min on ice in RIPA buffer supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF). Total cellular proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. Primary antibodies against ASF1A (1:1000, Cell Signaling Technology, Cat#2990), c-Myc (1:1000, Abcam, Cat#ab56), HES1 (1:1000, Cell Signaling Technology, Cat#11988), and actin (1:10000, Sigma) were incubated with the membranes at 4 °C overnight. Horseradish peroxidase-conjugated anti-rabbit and anti-mouse secondary antibodies (Jackson ImmunoResearch, USA) were diluted to 1:4000 and incubated with the respective membranes at room temperature for 50 min. Protein blots were visualized with enhanced chemiluminescence reaction (ECL+, Millipore, USA).
6
NF-κB Activation and Apoptosis Assay
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NF-κB oligonucleotide: Promega, Mannheim, Germany; [γ33P]ATP: Hartmann Analytic, Braunschweig, Germany; T4 polynucleotide kinase: New England Biolabs, Frankfurt, Germany; William's medium E, L-glutamine and DMEM: Biochrome, Berlin, Germany; LightCycler 480 Probes Master, penicillin-streptomycin, poly(dI-dC), cOmplete EDTA free protease inhibitor cocktail, PhosSTOP phosphatase inhibitor cocktail, 0.45 µm PVDF membrane and Cell Death Detection ELISAPLUS Kit: Roche, Mannheim, Germany; Quantikine Cytochrome c ELISA, recombinant IL-1β and recombinant TNFα: R&D Systems, Minneapolis, USA; RNeasy Plus Mini Kit and QuantiTect Reverse Transcription Kit: Qiagen, Hilden, Germany; primer and probes for quantitative RT-PCR 2: Sigma, Steinheim, Germany; actinomycin D (ActD): Enzo Life Sciences, Lörrach, Germany; p38 inhibitor RN3503 and JNK inhibitor SP600125: Prof. S. Laufer, University of Tübingen, Germany; Bradford Quick Start Dye: Bio-Rad Laboratories, Munich, Germany; ECL western blotting detection reagents, GE Healthcare, Little Chalfont, UK; pJNK, JNK, PARP, XIAP, A20/TNFAIP3, α-tubulin, pro-caspase-3 and cleaved caspase-3 antibodies: Cell Signaling, Danvers, USA; horseradish peroxidase conjugated anti-rabbit and anti-mouse secondary antibodies: Jackson Immuno Research, Newmarket, UK.
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