Total protein was extracted from BMMs and C3H10 cells and from the bones of mice used in the OVX experiment using a radioimmunoprecipitation assay lysis buffer (RIPA) (Sigma-Aldrich), and the protein concentration was measured using a bicinchoninic acid protein (BCA) assay kit (EMD Millipore, Bedford, MA, USA). Protein equivalents were separated using 10% SDS PAGE and transferred onto PVDF membranes (EMD Millipore). The membranes were blocked for an hour at room temperature with 5% skim milk in Tris-buffered saline with Tween 20. After incubating primary antibodies overnight at 4 °C, the membranes were nurtured with corresponding HRP conjugated secondary antibodies (Proteintech Group, Chicago, IL, USA.) The bands were visualized employing ECL Luminous Liquid (EMD Millipore). ImageJ software was used to compute the relative grey level of proteins (NIH). Anti-TRAIL was procured from Santa Cruz Bio (Santa Cruz, CA, USA). Anti-CTSK, TRAP, NFATc1, OPG, RUNX2, OCN, RANKL, COL1a, p-P65, P65, p-P38, P38, p-JNK, JNK, p-ERK, ERK, p-IKBα, IKBα, IKKα, IKKβ, and p-IKKα/β were all purchased from Cell Signaling Technology (Danvers, MA, USA).
P ikkα β
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom, China
P-IKKα/β is a laboratory antibody product that detects phosphorylated forms of the IKKα and IKKβ subunits of the IκB kinase complex. The IKK complex plays a central role in the activation of the NF-κB signaling pathway in response to various stimuli.
Automatically generated - may contain errors
Lab products found in correlation
P iκbα, by Cell Signaling Technology (51 mentions)
β actin, by Cell Signaling Technology (24 mentions)
P jnk, by Cell Signaling Technology (21 mentions)
P nf κb p65, by Cell Signaling Technology (20 mentions)
P erk, by Cell Signaling Technology (19 mentions)
Nf κb p65, by Cell Signaling Technology (19 mentions)
Gapdh, by Cell Signaling Technology (16 mentions)
P akt, by Cell Signaling Technology (15 mentions)
Ikkα β, by Cell Signaling Technology (12 mentions)
Pvdf membrane, by Merck Group (11 mentions)
P ikbα, by Cell Signaling Technology (9 mentions)
Bcl 2, by Cell Signaling Technology (9 mentions)
β actin, by Santa Cruz Biotechnology (9 mentions)
Erk1 2, by Cell Signaling Technology (8 mentions)
Cox 2, by Cell Signaling Technology (8 mentions)
Cleaved caspase 3, by Cell Signaling Technology (7 mentions)
P nf κb, by Cell Signaling Technology (7 mentions)
P erk1 2, by Cell Signaling Technology (6 mentions)
β actin, by Proteintech (6 mentions)
Nf κb, by Cell Signaling Technology (6 mentions)
116 protocols using p ikkα β
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of Signaling Pathways
3
Protein Extraction and Western Blot Analysis
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Proteins were extracted from cell lysis with the Radio Immunoprecipitation Assay (RIPA) buffer (Thermo Scientific, Carlsbad, California, USA) containing a protease inhibitor cocktail (Selleck, Huston, Texas, USA). BCA kit (Thermo Scientific, Carlsbad, California, USA) was used to quantify proteins and equal amounts was resolved by sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel and transferred to the PVDF membrane (Millipore, Billerica, MA). The membranes were blocked for 2h with 5% skim milk at room temperature and incubated with primary antibodies at 4°C overnight with gentle shake. The PVDF membrane was then incubated with appropriate secondary antibody for 1 h at room temperature. The bands in the membranes were then detected using the enhanced chemo luminescence substrate kit (Thermo Scientific, Carlsbad, California, USA) and scanned with Image J software. The antibodies used in our study including MMP9, CCND1, XIAP, TWIST1, IκBα, p-IκBα, p-IKKα/β, IKKα, IKKβ, N-Cadherin, E-Cadherin, Vimentin, Lamin B1 and GAPDH are purchased from Cell Signaling (CST, Danvers, MA, USA).
Subcellular fractions were prepared from ESCC cells with a Minute™ Cytoplasmic & Nuclear Extraction Kits (Invent biotech, Eden Prairie, USA) according to manufacturer’s instructions.
Subcellular fractions were prepared from ESCC cells with a Minute™ Cytoplasmic & Nuclear Extraction Kits (Invent biotech, Eden Prairie, USA) according to manufacturer’s instructions.
4
Comprehensive Protein Expression Analysis
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The anti-PARP, Survivin, Mcl-1, XIAP, Bcl-2, Bcl-XL, p15, p21, Cyclin D1, p-IKKα/β, IKKα, IKKβ, p-IκBα, IκBα, p-JNK, JNK, p65, Tubulin, RelB, Caspases-7 and -8, cleaved Caspase-7 and -8 antibodies were purchased from Cell Signaling (Cell Signaling Technology, Danvers, MA). The anti-Cyclin B1 antibody was purchased from Abnova (Abnova, Taipei, Taiwan). The anti-Cyclin E1 antibody was purchased from Zymed (Zymed, San Francisco, CA). The anti-p27 antibody was purchased from Becton Dickinson (BD, San Diego, CA). The anti-A20 antibody was purchased from eBioscience (eBioscience, San Diego, CA). The anti-histone H3 antibody was purchased from Abcam (Abcam, Cambridge, UK). The anti-Caspase-3 and cleaved Caspase-3 antibodies were purchased from Imagenex (Imagenes, San Diego, CA). The anti-β-actin antibody was purchased from Sigma (Sigma, St. Louis, MO).
The MDA-MB-468 and SW527 cell lysate was subjected to SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with diluted primary antibodies, horseradish peroxidase (HRP) conjugated secondary antibodies (Jackson ImmunoResearch Laboratory, West Grove, PA), and Western Lighting Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, Shelton, CT) and were visualized on an ImageQuant LAS4000 Biomolecular imager to determine the expression levels of specific proteins.
The MDA-MB-468 and SW527 cell lysate was subjected to SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with diluted primary antibodies, horseradish peroxidase (HRP) conjugated secondary antibodies (Jackson ImmunoResearch Laboratory, West Grove, PA), and Western Lighting Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, Shelton, CT) and were visualized on an ImageQuant LAS4000 Biomolecular imager to determine the expression levels of specific proteins.
5
Melatonin and Epirubicin Anticancer Effects
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Melatonin was purchased from J&K, Chemical Ltd., and epirubicin was purchased from Pfizer, pharmaceutical Ltd. Melatonin and epirubicin were dissolved in dimethyl sulphoxide (DMSO) and made into stock solution before adding into the complete culture medium. The maximum concentration of DMSO in the culture medium did not exceed 0.1%. The primary antibodies for GAPDH, LaminB1, and P-glycoprotein were purchased from Proteintech (Proteintech, Inc., USA). The antibodies for Bcl-2, cleaved-PARP, IKKβ, p-IKKα/β, NF-κB P50, and P65 were purchased from Cell Signaling Technology (Cell Signaling Technology, Inc., USA). The antibody for cytochrome c was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
6
Costunolide Attenuates Inflammatory Response
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Costunolide (C15H20O2; molecular weight 232.32), abbreviated as CTL, with purity of 99.84% confirmed by MS and H1NMR data, was purchased from MedChemExpress LLC (Shanghai, China). LPS was from Sigma-Aldrich (St. Louis, MO, USA). Fatty acid-free bovine serum albumin (BSA) was obtained from Equitech-Bio (Kerrville, TX, USA). Antibodies against iNOS, COX2, GAPDH, p-AKT, AKT, p-IKKα/β, IKKβ, p-IκB-α, IκB-α, p-NF-κB p65, NF-κB p65, CDK2, Histone H3, and α-Tubulin were purchased from Cell Signaling Technology (Beverly, MA, USA). Recombinant human CDK2 protein was acquired from Sino Biological (Beijing, China).
7
Western Blotting of Glioma Signaling Proteins
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Western blotting was performed as previously described 17 (link). Briefly, the total proteins of glioma cells or tissues were isolated using a total cell protein extraction kit (KeyGen Biotechnology, Nanjing, China). Protein lysates were transferred onto PVDF membranes after electrophoresis and blocked with 2% bovine serum albumin (KeyGen Biotechnology). The primary antibodies against SLC39A7 (1:1000; Abcam), TNF-α (1:1000; Abcam), p-p65 (1:1000; Cell Signaling Technology, Danvers, MA, USA), p65 (1:1000; Cell Signaling Technology), p-IκBα (1:1000; Cell Signaling Technology), IκBα (1:1000; Cell Signaling Technology), p-IKKα/β (1:1000; Cell Signaling Technology), IKKα (1:500; Cell Signaling Technology), IKKβ (1:500; Cell Signaling Technology) and β-actin (1:2000; ProteinTech, Chicago, IL, USA) were incubated at 4 °C overnight. After secondary antibody (ProteinTech) incubation, the bands were detected using a chemiluminescence ECL kit (Beyotime Biotechnology, Beijing, China) and quantified by Image J software (National Institutes of Health, Bethesda, MD, USA).
8
Murine Macrophage Culture and Stimulation
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RAW 264.7 murine macrophages were obtained from the American Type Culture Collection (Manassas, VA, USA) and were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin G, 100 μg/ml streptomycin) at 37℃ in a humidified incubator containing 5% CO2 and 95% air. DMEM and PBS were purchased from Cellgro Mediatech Inc. (Herndon, VA, USA). Antibodies for p65, Lamin B, and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for iNOS, IκBα, p-p65, p-IKKα/β, IKKα, IKKβ, p-ERK, ERK, p-p38, p38, p-JNK, and JNK were purchased from Cell Signaling Technology (Beverly, MA, USA). Lipopolysaccharide (LPS, Escherichia coli 0127:B8) and horseradish peroxidase (HRP)-conjugated anti-rabbit or goat antibodies were supplied by Sigma (St. Louis, MO, USA). Greiss reagent and luciferase assay kits were obtained from Promega (Madison, CA, USA). ELISA kits for TNF-α, IL-6, and IL-1β were purchased from R&D systems (Minneapolis, MN, USA). iNOS, TNF-α, IL-6, IL-1β, and β-actin oligonucleotide primers were purchased from Bioneer (Seoul, Korea).
9
Antibody Generation and Immunoblotting
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Western blotting and immunoprecipitations were performed using the following primary homemade antibody: a monoclonal antibody against Morgana (P1PP0) was generated in our laboratory using GST-morgana fusion protein as an antigen20 (link). Other commercially antibodies were used: Vinculin (Sigma, SAB4200080, 1:5000), CHORDC1 (Sigma, HPA041040, 1:1000), MMP9 (Abcam, ab76003, 1:1000), MMP2 (Santa Cruz, 8835, 1:500), HSP90 (Santa Cruz, 13119, 1:1000), P-IkBα Ser32-36 (Santa Cruz, 8404, 1:500), P-IkBα Tyr42 (Abcam, ab24783, 1:1000), IkBα (Cell Signaling, 4814, 1:1000), IKKα (Cell Signaling, 11930, 1:1000), IKKα (Cell Signaling, 2682, 1:100 for co-immunoprecipitation experiments), IKKβ (Cell Signaling, 8943, 1:1000), IKKγ (Santa Cruz, 8032, 1:500), αTubulin (Sigma, T5168, 1:8000), MBP (Cell Signaling, 2396, 1:1000), TNF-R1 (Santa Cruz, 8436, 1:500), TAK-1 (Santa Cruz, 166562, 1:500), p-AKT (Cell Signaling, 9271, 1:1000), AKT (Cell Signaling, 4691, 1:1000), CDC37 (Santa Cruz, 5617, 1:500), P-IKKα/β (Cell Signaling, 2697, 1:1000).
TNFα (300-01A and 315-01A) was purchased from Peprotech, the IKKβ inhibitor PS1145 (P6624), ROCK inhibitor Y27632 (Y0503), the PTEN inhibitor VO-OHpic (V8639), and the AKT inhibitor GSK69093 (SML0428) were from Sigma. BEZ235 (S1009) was purchased from Selleckchem.
TNFα (300-01A and 315-01A) was purchased from Peprotech, the IKKβ inhibitor PS1145 (P6624), ROCK inhibitor Y27632 (Y0503), the PTEN inhibitor VO-OHpic (V8639), and the AKT inhibitor GSK69093 (SML0428) were from Sigma. BEZ235 (S1009) was purchased from Selleckchem.
10
Western Blot Analysis of Protein Targets
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