Proteins from cells and cerebral microvessels were obtained with lysis buffer (Thermo Scientific, FL) containing protease inhibitors. The proteins were subjected to electrophoresis and transferred onto nitrocellulose membranes. The membranes were blocked by incubating with 5% dry milk for 1 hr, and then incubated with primary antibodies: against NFκB (1:1000; Cell signaling, Danvers, MA), Inhibitor of κBα (IκBα) (1:1000; Cell signaling, Danvers, MA), at 4°C overnight. After being washed thoroughly, membranes were incubated with horseradish peroxidase (HRP) conjugated IgG (1:40000; Jackson ImmunoResearch Labs, INC. PA) for 1 hr at RT. β-actin (1:4000; Sigma-Aldrich, St. Louis, MO) was used to normalize protein loading.
Horseradish peroxidase hrp conjugated igg
Manufactured by Jackson ImmunoResearch
Sourced in United States
Horseradish peroxidase (HRP) conjugated IgG is a laboratory reagent that consists of horseradish peroxidase enzyme covalently linked to immunoglobulin G (IgG) antibodies. This conjugate can be used as a detection or labeling agent in various immunoassay techniques, such as enzyme-linked immunosorbent assay (ELISA).
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (3 mentions)
Lysis buffer, by Thermo Fisher Scientific (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Nf κb, by Cell Signaling Technology (1 mentions)
Anti sox2, by Cell Signaling Technology (1 mentions)
Immobilon ecl chemiluminescence hrp substrate, by Merck Group (1 mentions)
Protease inhibitor, by Keygen Biotech (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Enhanced chemiluminescence detection kit, by Merck Group (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Rapamycin, by Merck Group (1 mentions)
Pf 4708671, by Merck Group (1 mentions)
Hif 1α, by Horizon Discovery (1 mentions)
Nf κb p65, by Santa Cruz Biotechnology (1 mentions)
P70s6k1, by Cell Signaling Technology (1 mentions)
Sk n mc, by Korean Cell Line Bank (1 mentions)
Flotillin 2, by Santa Cruz Biotechnology (1 mentions)
P nf κb p65 ser536, by Santa Cruz Biotechnology (1 mentions)
Gpr40, by Abcam (1 mentions)
Gw9508, by Merck Group (1 mentions)
9 protocols using horseradish peroxidase hrp conjugated igg
1
Western Blot Analysis of NFκB Pathway
2
Quantitative Western Blot Analysis
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Cells ready for Western blotting analysis were harvested and washed with cold PBS twice, then lysed on ice in RIPA buffer (1 × PBS, 1% NP-40, 0.1% sodium dodecylsulfate (SDS), 5 mM EDTA, 0.5% sodium deoxycholate, and 1 mM sodium orthovanadate) that contained 100 μg/mL phenylmethylsul-fonylsuoride and protease inhibitors (KeyGen, Nanjing, China).
Approximately 50 μg of protein from each sample was separated using a 10% SDS-polyacrylamide gel, electrotransfered to polyvinylidene fluoride(PVDF) membranes and blocked in 5% nonfat dry milk in Tris-buffered saline, pH 7.5 (100 mM NaCl, 50 mM Tris, and 0.1% Tween-20). The transferred membranes were incubated with anti-SOX2 (Cell Signaling Technology, USA) and anti-β-actin primary antibodies (Beyotime, Jiangsu, China) overnight at 4°C, followed by incubation with horseradish peroxidase(HRP) conjugated IgG(JacksonImmunoResearch, USA). Proteins were detected by Quantity-one software (Bio-Rad, Laboratories, Inc, USA) using Immobilon ECL Chemiluminescence HRP Substrate (Millipore, Merck, USA).
Approximately 50 μg of protein from each sample was separated using a 10% SDS-polyacrylamide gel, electrotransfered to polyvinylidene fluoride(PVDF) membranes and blocked in 5% nonfat dry milk in Tris-buffered saline, pH 7.5 (100 mM NaCl, 50 mM Tris, and 0.1% Tween-20). The transferred membranes were incubated with anti-SOX2 (Cell Signaling Technology, USA) and anti-β-actin primary antibodies (Beyotime, Jiangsu, China) overnight at 4°C, followed by incubation with horseradish peroxidase(HRP) conjugated IgG(JacksonImmunoResearch, USA). Proteins were detected by Quantity-one software (Bio-Rad, Laboratories, Inc, USA) using Immobilon ECL Chemiluminescence HRP Substrate (Millipore, Merck, USA).
3
Western Blot Analysis of miR-125 and Afa Effects
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AGS (8 × 105) cells were seeded in 6 cm plates. After overnight incubation, the cells were treated with different miR-125 (100 nM) and/or Afa (300 nM)-loaded formulations. After 24 h of incubation, the cells were lysed. The proteins were quantified using BCA protein assay (Thermo Fisher, Waltham, MA, USA). Protein (30 µg) in the volume of 10 µL was loaded for each band, separated via 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE), and shifted onto PVDF membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked and incubated with primary antibodies from Cell Signaling (USA) or Abcam (UK) overnight at 4 °C. After conjugating with horseradish peroxidase (HRP)-conjugated Ig G (Jackson), the membranes were monitored using an enhanced chemiluminescence detection kit (Millipore, Billerica, MA, USA). Names and concentrations of antibodies have been demonstrated in Table S1 .
4
Neuroblastoma Cell Line: Akt-mTOR Signaling
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The human neuroblastoma cell line SK-N-MC was obtained from Korean Cell Line Bank (Seoul, Korea). The antibodies of p-Akt (Thr308), p-Akt (Ser473), Akt, mTOR, Caveolin-1, Flotillin-2, p-Tau (Ser396), Tau, p-NF-κB p65 (Ser536), NF-κB p65, Lamin A/C, CBP and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibodies of p-mTOR (Ser2448), p-p70S6K1 (Thr389) and p70S6K1, were acquired from Cell Signaling Technology (Beverly, MA, USA). Aβ, BACE1, HIF-1α and GPR40 antibodies were obtained from Abcam (Cambridge, MA, USA). The C99 antibody was purchased from EMD Millipore (Darmstadt, Germany). Horse radish peroxidase (HRP)-conjugated IgG was obtained from Jackson Immunoresearch (West Groove, PA, USA). PA, BSA, GW9508, ionomycin, PF4708671, LY294002 and rapamycin were purchased from Sigma Chemical Company (St. Louis, MO, USA). The Akt inhibitor, GW1100 and SN50 used here were purchased from Calbiochem (La Jolla, CA, USA). siRNAs for GPR40, GPR120, APP, BACE1, HIF-1α and non-targeting were obtained from Dharmacon (Lafayette, CO, USA).
5
Hypoxia Signaling Pathway Analysis
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CoCl2 (#232696, Sigma‐Aldrich) and verteporfin (VP; #5305, Tocris Bioscience) were purchased from Sigma‐Aldrich and Bio‐Techne, respectively. The antibodies were used consist of HIF‐1α (#127309, GenTex), YAP (#H00010413‐M01, Abnova), phospho‐Paxillin Tyr118 (#69363s, BD Biosciences), Paxillin (#611051, BD Biosciences), Hoechst 33342 (#H1399, Invitrogen), GAPDH (#100118, GenTex). The secondary antibody recognized the primary antibody consisting of AlexaFluor® 488 and AlexaFluor® 594 antibodies (#ab150077, #ab150113 and #ab150116, Abcam). The secondary antibody used for western blotting was horse radish peroxidase (HRP) conjugated IgG (#115‐035‐003 and 115‐035‐003, Jackson ImmunoResearch Laboratories).
6
Epothilone Analog Evaluation in Cells
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Novel epothilone analogs were obtained from Biostar Technologies (Beijing, China). All the cell culture reagents were purchased from Invitrogen (San Diego, USA). MTT, Triton X-100, β-tubulin antibody, Hoechst 33342, and Matrigel were purchased from Sigma-Aldrich (St. Louis, USA). Rac1, Cdc42 and p-PAK1 primary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, USA). p-ERK1/2, p-JNK, p-p38, p-Akt, PAK1, ERK1/2, JNK, Akt and p38 antibodies were purchased from Abcam (Cambridge, UK). Horseradish peroxidase (HRP)-conjugated IgG and FITC-labeled mouse IgG were acquired from Jackson ImmunoResearch Laboratories (West Grove, USA). bFGF and fibronectin were obtained from R&D systems (Minneapolis, USA). The ECL regent was purchased from GE Healthcare Life Science (Pittsburgh, USA). The polyvinylidene fluoride (PVDF) membrane was acquired from Millipore (Massachusetts, USA). Superscript II reverse transcriptase was the product of Takara (Dalian, China). UTD2 was initially dissolved in dimethyl sulfoxide (DMSO) as a stock solution and subsequently diluted to the desired experimental concentrations with culture media.
7
Western Blot Analysis of Cardiomyocyte Proteins
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After different treatments, proteins from H9c2 cells were obtained with lysis buffer (Thermo Scientific, FL) containing protease inhibitor. The proteins were subjected to electrophoresis and transferred onto nitrocellulose membranes. The membranes were blocked by incubating with 5% dry milk for 1 h, and then incubated with primary antibodies: against β-MHC (1∶1000; Sigma-Aldrich, St. Louis, MO), Akt (1∶500; Cell Signaling Technology, MA), p-Akt (Thr-308, 1∶500; Cell Signaling Technology, MA), eNOS (1∶500; Cell Signaling Technology, MA) or p-eNOS (ser-1177, 1∶500; Cell Signaling Technology, MA), at 4°C overnight. β-actin (1∶4000; Sigma-Aldrich, St. Louis, MO) was used to normalize protein loading. After being washed thoroughly, membranes were incubated with horseradish peroxidase (HRP) conjugated IgG (1∶40000; Jackson ImmunoResearch Labs, INC. PA) for 1 h at RT. Blots were then developed with enhanced chemiluminescence developing solutions and quantified [27] (link).
8
Tim-3 Protein Expression Quantification
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Proteins were extracted from cells with lysis buffer containing protease inhibitor (Roche Diagnostics). Proteins were separated by SDS-PAGE and transferred onto PVDF membrane. The PVDF membrane was blocked with 5% nonfat milk in 1x Tris-buffered saline with Tween-20 (TBST, pH 7.6) at room temperature (RT) for 1 h and then incubated with primary antibody against Tim-3 (1 : 200; Santa Cruz) at 4°C overnight. β-actin (1 : 40000; Sigma) was used to normalize protein loading. After being washed with TBST thrice, membranes were incubated with horseradish peroxidase- (HRP-) conjugated IgG (1 : 40000, Jackson Lab) for 1 hour at RT. Blots were then developed with enhanced chemiluminescence developing solutions and quantified.
9
Evaluating Endothelial Cell Function
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Adenovirus pAD/DDAHII and pAD/DDAHII-shRNA were from GeneChem Co., Ltd. (Shanghai, China). Iron dextran (iron-D), dextran (Dex), phenylephrine (PE), sodium nitroprusside (SNP), acetylcholine (Ach), L-arginine (L-Arg), Edaravone (Eda), N-nitro-l-arginine methyl ester (l-NAME), and ciclosporin A (CsA) were purchased from Sigma-Aldrich (cat. nos. D8517, D9885, P1240000, PHR1423, PHR1546, A5006, M70800, N5751, and C1832, respectively, St. Louis, MO, USA). Antibodies directed against DDAHII, eNOS, eNOS phospho-S1177, cytochrome c (cyt c), COX4, and β-actin were purchased from Abcam (cat. no. ab1383, ab5589, ab184154, ab16381, ab13575, and ab8229, respectively, Cambridge, UK). Horseradish peroxidase- (HRP-) conjugated IgG was from Jackson ImmunoResearch (cat. no. 107-035-142, West Grove, PA, USA).
Human umbilical vein endothelial cells (HUVECs) were purchased from the China Infrastructure of Cell Line Resources (Shanghai, China). Male C57BL/6J mice, 8-10 weeks old, weighing 20-22 g, were provided by the Animal Center of Nanchang University (Nanchang, China).
All experiments were performed following the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996) and were approved by the Ethics Committee of Nanchang University (Nanchang, China) (No. 2017-0306 (in vitro) and 2017-0305 (in vivo)).
Human umbilical vein endothelial cells (HUVECs) were purchased from the China Infrastructure of Cell Line Resources (Shanghai, China). Male C57BL/6J mice, 8-10 weeks old, weighing 20-22 g, were provided by the Animal Center of Nanchang University (Nanchang, China).
All experiments were performed following the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996) and were approved by the Ethics Committee of Nanchang University (Nanchang, China) (No. 2017-0306 (in vitro) and 2017-0305 (in vivo)).
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