BDNF, leupeptin, aprotinin, phenyl-methylsulfonyl fluoride, pepstatin A, soybean trypsin inhibitor, NaF, sodium vanadate, glycerophosphate, 2-mercaptoethanol, NMDA, glycine, Tween 20, NP-40, and Histopaque-1077 were from Sigma. Anti-TrkB (SC-8316), -pY-Trk (SC-8058), -phospholipase C-γ1 (SC-7290), -N-Shc (SC-365598), -NR1 (SC-9058), -TNFα (SC-52746), -IL-6 (SC-57315), -IL-1β (SC-57315), -Arc (SC-365736), - BDNF/proBDNF (SC-2098), –NT3 (SC-547), -NT4 (SC-545), -β-actin (SC-47778) were from Santa Cruz Biotechnology. FNDC-5/irisin (36–335) was from ProSci (Poway, CA, USA). Seize-X immunoprecipitation kit, antigen elution buffer, Bind NeutrAvidin, high binding capacity coated 96-well plates, and West Pico chemiluminescent reagents were from Pierce-Endogen (Rockford, IL, USA). Bradford reagent, SDS-PAGE reagents, and pre-stained molecular weight markers were from Bio-Rad. Protease inhibitors (EDTA-free) and protein phosphatase inhibitor tablets were from Roche (Basel, Switzerland). BDNF was reconstituted according to the manufacturer’s instruction. To avoid freezing damage, 10% glycerol was added for a final concentration of 10 ng/μl BDNF and stored in 80°C until use. All other test agents were made fresh according to the manufacturer’s recommendation. The DMSO concentration in the incubation medium was 1%.
Sds page reagents
Manufactured by Bio-Rad
Sourced in United States, Italy
SDS-PAGE reagents are a set of chemicals used to perform sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a widely used technique in biochemistry and molecular biology for the separation and analysis of proteins based on their molecular weight.
Automatically generated - may contain errors
Lab products found in correlation
Protein standard, by Bio-Rad (2 mentions)
Sds sample buffer, by Bio-Rad (2 mentions)
Anti β actin antibody, by Merck Group (2 mentions)
Hanks balanced salt solution (hbss), by Merck Group (2 mentions)
Rc dc protein assay, by Bio-Rad (2 mentions)
Rpmi 1640, by Merck Group (2 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
L glutamine, by Merck Group (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Merck Group (2 mentions)
Prestained molecular weight markers, by Bio-Rad (1 mentions)
Brain derived neurotrophic factor (bdnf), by Merck Group (1 mentions)
Bdnf probdnf, by Santa Cruz Biotechnology (1 mentions)
Il 1β, by Santa Cruz Biotechnology (1 mentions)
Tnf α, by Santa Cruz Biotechnology (1 mentions)
Np 40, by Merck Group (1 mentions)
Pepstatin a, by Merck Group (1 mentions)
Anti trkb, by Santa Cruz Biotechnology (1 mentions)
Tween 20, by Merck Group (1 mentions)
14 protocols using sds page reagents
1
BDNF Signaling Protein Detection
2
Neutrophil Signaling Pathway Analysis
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The N-fMLP peptide was synthesized and HPLC-purified by PRIMM (Milan, Italy). SDS-PAGE reagents were purchased from Bio-Rad (Hercules, CA, USA). Protein A/G Plus, anti-Flk1, anti-p-Flk1 (Tyr951), anti-p-Flk1 (Tyr996), anti-p-Flk1 (Tyr1175), anti-p-Flk1 (Tyr1214), anti-p-Tyr, anti-p47phox, anti-p22phox, anti-p-PLCγ1 (Y783), anti-PLCγ1, anti-PKCα, anti-PKCβII, anti-PKCζ, anti-PKCδ, anti-tubulin, anti-mouse, and anti-rabbit were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p-PI3K (p85) and anti-p-Akt (Ser473) were from Cell Signaling Technology (Danvers, MA, USA). Anti-p-Ser, p22phox siRNA (SI03078523), and scramble control siRNA (SI03650318) were from Qiagen (Hiden, Germany). FPR1 siRNA (L-005140-00) and scramble control (D-001810-10) were purchased from Dharmacon (Lafayette, CO, USA). Protein A-horseradish peroxidase was from Amersham Pharmacia Biotech (Little Chalfont, Buckinghamshire, UK). Pertussis toxin (PTX), apocynin, wortmannin, and LY294002 were from Sigma (St. Louis, MO, USA).
3
Purification and Characterization of Anti-Cocaine mAb
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The generation, production, and purification of the h2E2 anti-cocaine monoclonal antibody by the manufacturer, Catalent, was previously described [8 (link)]. Fluorescein isothiocyanate (FITC) was purchased from Invitrogen (isomer 1, cat #F1907). Sephadex G-50-150 was purchased from Sigma. Fab and Fc fragments of this mAb were generated by proteolysis using Endo-Lys-C immobilized on agarose beads (SignalChem, cat #L585-31AN-1). A 10 mM stock solution of cocaine in distilled water was made from solid cocaine HCl as described [9 (link)]. PBS buffer was made by diluting PBS buffer concentrate (10X) purchased from Cambrex (BioWhittaker, without calcium or magnesium, catalog number 17-517Q). The Applied Biosystems 48 well RT PCR plates (cat. 4375816) and the MicroAmp plate sealing optical adhesive transparent film (cat. 4375928) used in the StepOne RT PCR instrument used to perform the DSF analyses were purchased from ThermoFisher Scientific. Electrophoresis chemicals and SDS-PAGE reagents were purchased from BioRad. The SDS-PAGE gel protein standards were from SMOBIO (cat. PM1600).
4
Immunoblotting and Immunoprecipitation in HEK-3KO Cells
5
Immunoblotting of Lysosomal and Signaling Proteins
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Mouse anti-LAMP1 monoclonal antibody (555798) was purchased from BD Biosciences; mouse anti-diphosphorylated ERK1/2 monoclonal antibody (M8159) was from Sigma-Aldrich; rabbit anti-ERK1/2 polyclonal antibody (V114A) was from Promega; mouse anti-β-actin monoclonal antibody (G043) was from Abm; mouse anti-γ-tubulin antibody (T6557) was from Sigma-Aldrich; goat anti-mouse IgG polyclonal antibody conjugated to horseradish peroxidase (HRP) (sc-2031) and goat anti-rabbit IgG-HRP polyclonal antibody (sc-3837) were from Santa Cruz Biotechnology; goat anti-mouse IgG-TRITC antibody (T5393) was from Sigma-Aldrich; BSA (A7906) was from Sigma-Aldrich; SDS-PAGE reagents were from Bio-Rad; fetal bovine serum (FBS) was from Gibco; LysoTracker (L7528) was from Thermo Fisher Scientific; fibronectin (F2006) was from Sigma-Aldrich; FGF2 was from PeproTech; and Alcian blue dye (74240) was from EuroDiagnostica. The recombinant NK1 fragment of HGF was produced using yeast Pichia pastoris expression system and purified with heparin affinity chromatography as previously described.39 (link)
6
Endoplasmic Reticulum Stress Pathway
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Antibody anti-β-actin, BSA, FCS, HBSS, l-glutamine, penicillin-streptomycin, PBS, propidium iodide (PI), Q, RNAse, RPMI-1640, and tunicamycin were from Sigma-Aldrich (St. Louis, MO, USA). Antibodies anti-BiP, -caspase 3, -CHOP, -Hsp70, -Hsp90, -IRE1α, and -PARP were from Cell Signaling Technology (Beverly, MA, USA). Horseradish peroxidase- (HRP-) conjugated anti-rabbit- and anti-mouse-immunoglobulin antibodies, enhanced chemiluminescence (ECL) reagents, and Hyperfilm-ECL film were from Amersham (Arlington Heights, IL, USA). Lipofectamine RNAiMAX and OPTI-MEM medium, small interfering- (siRNA-) IRE1, and relative scrambled siRNA (Ambion) were from Life Technologies (Invitrogen, San Giuliano Milanese, Italy). 2-Phenylethynesulfonamide (PES) and 4μ8c were from Merck (INALCO, Milan, Italy). RC-DC protein assay, SDS-sample buffer, protein standard, SDS-PAGE reagents, and polyvinylidene difluoride (PVDF) membranes were from Bio-Rad Laboratories (Segrate, Italy). Antibody anti-tubulin, siRNA-Hsp70, and scrambled siRNA were purchased from Santa Cruz Biotechnology (Tebu-Bio, Magenta, Italy). Thapsigargin and z-VAD.fmk were from Calbiochem (San Diego, CA, USA). Other reagents were of the highest purity and purchased from Bio-Rad, Invitrogen, or Sigma.
7
Immunofluorescence Staining and Cellular Analysis
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4′,6-diamidino-2-phenylindole (DAPI) (D9542, Sigma-Aldrich); SDS-PAGE reagents (Bio-Rad Laboratories, Hercules, CA, USA); PD98059 [2-(2′-amino-3′-methoxyphenyl)-oxanaphthalen-4-one] (513,000, Calbiochem, La Jolla, CA, USA); AG1478 [N-(3-chlorophenyl)−6,7-dimethoxy-4-quinazolinamine], (T4182, Sigma-Aldrich); puromycin, streptomycin and penicillin, and bovine serum albumin (BSA) (Sigma-Aldrich); fetal bovine serum (FBS) (GIBCO, Karlsruhe, Germany); LysoTracker (L7528, Thermo Fisher Scientific, Fremont, CA, USA), phalloidin (P1951, Sigma-Aldrich); lectin from Triticum vulgaris (wheat) FITC conjugate (L4895, Sigma-Aldrich); IBAfect reagent (7-2005-050, IBA Lifesciences, Goettingen, Germany); 4-methylumbelliferyl-N-acetyl-α-D-glucosaminide (474,500, Calbiochem); Trizol reagent (15596026, Invitrogen, Carlsbad, CA, USA).
8
ER Stress and Autophagy Regulation
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Antibody anti-SQSTM1/p62 was from BD Transduction Laboratories (San Jose, CA, USA). Antibodies anti-BiP, -CHOP, phoshorylated-eIF2α (p-eIF2α), total–eIF2α (t-eIF2α), -IRE1α, -LC3I/II and horseradish peroxidase (HRP)-conjugated anti-rabbit- and anti-mouse-immunoglobulin antibodies were from Cell Signaling Technology (Danvers, MA, USA). Antibody anti-β actin, bafilomycin, bovine serum albumin (BSA), chloroquine, HBSS, l-glutamine, 3-methyladenine, penicillin-streptomycin, phosphate buffered saline (PBS), PI, RNAse, and RPMI-1640 were from Sigma-Aldrich (St. Louis, MO, USA). Fetal calf serum (FCS) (GIBCO), LysoTracker-Red, Lipofectamine RNAiMAX and OPTI-MEM medium (GIBCO), siRNA-IRE1 (Ambion), siRNA-PERK (Ambion) and scrambled siRNA (Ambion) were from Life Technologies (Invitrogen, San Giuliano Milanese, Italy). RC-DC protein assay, SDS-sample buffer, protein standard, SDS-PAGE reagents and polyvinylidene difluoride membranes were from Bio-Rad Laboratories (Segrate, Italy). ECL fast femto was from Società Italiana Chimici (Rome, Italy). TN, 4μ8c and GSK2606414 were from Calbiochem (San Diego, CA, USA).
9
Carbon Tetrachloride Protein Assay IHC
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Carbon tetrachloride reagent grade 99.9% (319,961) was obtained from Sigma- Aldrich. BCA Protein Assay kit (23,227) was provided from Pierce Thermo Fisher Scientific. SDS-PAGE reagents were obtained from Bio-Rad. The reagents used for IHC, Avidin/Biotin Blocking Kit (SP-2001) and M.O.M. elite peroxidase kit (BMK-2202) were purchased from Vector Laboratories.
10
VEGF-Induced Signaling Pathway Analysis
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Tissue culture reagents, unless otherwise specified, were obtained from Biowhittaker (Walkersville, MD). Human recombinant VEGF, bFGF and epidermal growth factor (EGF) were purchased from PrepoTech (Rocky Hill, NJ). Sugen 5416 was obtained from Cayman Chemical Company (Ann Arbor, MI) and human VEGF neutralizing antibody was purchased from R&D Systems Europe (Abingdon, UK). Inhibitors LY294002, PD98059, and SB203580 were purchased from Cell Signaling Technology, Inc. (Bioke, Leiden, NL). Antibodies against Akt, Phospho-Akt (S473), p44/42 MAPK (ERK1/2), Phospho-p44/42 MAPK (ERK1/2), p38 MAPK, and Phospho- p38 MAPK (Thr180/Tyr182) were from Cell Signaling and mouse anti-human β actin monoclonal antibody was from Abcam (Cambridge, UK). Gelatin was obtained from Sigma-Aldrich and Fibronectin from Roche Diagnostics. SDS-PAGE reagents were obtained from BioRad (Hercules, CA). TaqMan Gene Expression assays were purchased from Applied Biosystems (Carlsbad, CA), TRIzol reagent and First Strand cDNA synthesis kits were purchased from Invitrogen (Carlsbad, CA). All other reagents were from Sigma-Aldrich Chemie B.V. (Zwijndrecht, NL), unless stated otherwise.
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