All primary human vascular endothelial cells including HAEC, HCAEC, HDMEC, HLMEC and HUVEC were acquired from Lonza Walkersville Inc. (Walkersville, MD) and cultured in EGM or EGM2 medium according to the manufacturer’s instruction. In all the experiments, cells were used within five passages. The human acute monocytic leukemia cell line THP-1 was obtained from American Type Culture Collection (ATCC) and was grown in RPMI 1640 containing 10% fetal bovine serum. Other cell lines including CHO, Cos-7, NIH3T3, 293 T, HeLa, Jurkat, RAW264.7 and U937 were purchased from ATCC and cultured in DMEM supplemented with 10% fetal bovine serum. Human recombinant TNFα and LPS were purchased from Sigma (Saint Louis, MO). VCAM-1 (sc-13160), ICAM-1 (sc-1511-R), E-selectin (sc-14011), IKKα/β (sc-7607), and actin (sc-1616) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-JNK, JNK, phospho-IKK, IKK, phosphor-ERK1/2, ERK1/2, phosphor-p38 and p38 antibodies were purchased from Cell Signaling Technology.
Phospho ikk
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-IKK is a laboratory reagent used for the detection and quantification of phosphorylated IKK (Inhibitor of kappa B kinase) proteins. IKK is a key component of the NF-κB signaling pathway, which regulates various cellular processes. The Phospho-IKK product provides a tool for researchers to analyze the activation status of the IKK complex in their experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Phospho jnk, by Cell Signaling Technology (6 mentions)
Phospho p38, by Cell Signaling Technology (5 mentions)
Phospho p65, by Cell Signaling Technology (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Phospho nf κb, by Cell Signaling Technology (3 mentions)
Phospho iκb, by Cell Signaling Technology (3 mentions)
Phospho akt, by Cell Signaling Technology (3 mentions)
Erk1 2, by Cell Signaling Technology (2 mentions)
Phospho erk, by Cell Signaling Technology (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Phosphor p38, by Cell Signaling Technology (1 mentions)
Hdmec, by Lonza (1 mentions)
Phosphor erk1 2, by Cell Signaling Technology (1 mentions)
Hcaec, by Lonza (1 mentions)
Hlmec, by Lonza (1 mentions)
Actin sc 1616, by Santa Cruz Biotechnology (1 mentions)
Raw264, by American Type Culture Collection (1 mentions)
Jurkat, by American Type Culture Collection (1 mentions)
Cos 7, by American Type Culture Collection (1 mentions)
11 protocols using phospho ikk
1
Vascular Endothelial Cell Isolation and Culture
2
Dicentrine Modulates Cell Signaling Pathways
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Dulbecco’s modified Eagle’s medium (DMEM), penicilin-streptomycin, and trypsin-ethylenediaminetetra acetic acid (EDTA) were purchased from GIBCO-BRL (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT, USA). Gelatin, PI, and MTT were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies specific to caspase-3, caspase-8, caspase-9, cFILP, cIAP-2, Bcl-XL, COX-2, phospho-NF-κB, phospho-c-Jun, c-Jun, phospho-IKK, phospho-IκB, phospho-p38, p38, phospho-JNK, JNK, phospho-ERK1/2, ERK1/2, phospho-AKT, AKT, phospho-TAK, TAK, and RIP were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). NF-κB, PARP, MT1-MMP, uPAR, ICAM-1, and VEGF were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cyclin D1 was purchased from Milipore. The Can Get Signal® Immunoreaction Enhancer Solution was purchased from Toyobo (Osaka, Japan). Matrigel was purchased from Becton Dickinson (Bedford, MA, USA). Dicentrine was ordered from Chengdu Biopurify Phytochemicals Ltd. (Sichuan, China).
3
Western Blot Analysis of Immune Signaling
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Protein samples were extracted using RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors (Thermo Fisher Scientific, Waltham, MA, USA). 20–40 μg of the cell lysate, which were determined by the BCA protein assay, were separated on 12% SDS-PAGE gels and electrophoretically transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). After blocking, the membranes were incubated overnight at 4°C with primary antibodies to CD32, CD64 (R&D Systems, Minneapolis, MN,USA), p38, phospho-p38(Cell Signaling, US), IκBα, phospho-IκBα, phospho-IKK, phospho-p65, p65, and TWIST (Cell Signaling Technology, Danvers, MA,USA), and house-keeping protein controls such as β-actin, α-tubulin or laminin B1(BD Pharmingen, San Diego, CA, USA). After washing, the membranes were incubated with HRP-conjugated secondary antibodies (R&D Systems) for 1 h and detected by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech, USA) reaction. Positive immunoreactive bands were quantified and normalized by β-actin using κκ The Image G program (NIH).
4
Protein Expression Analysis in Lung Tissue
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Whole protein from cell lysates and lung tissues was extracted according to manufacturer's instructions (Keygene, China). Protein samples were separated on 10% SDS‐PAGE gels, and then, the proteins were transferred to PVDF membranes (Merck Millipore, Germany), blocked with 5% nonfat milk in TBST and incubated with primary antibody at room temperature for 4 hr or overnight. Then, membranes were incubated with HRP‐conjugated secondary antibody for one hour, and protein expression was detected using ECL (Merck Millipore, Germany). Primary antibodies against P16, P21, αSMA, and Collagen1α were purchased from Abcam (United Kingdom). Antibodies against PTEN, IKK, phospho‐IKK, IκB, phospho‐IκB, NF‐κB, and phospho‐NF‐κB were purchased from Cell Signaling Technology (USA).
5
Western Blot Analysis of Protein Signaling
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After two washes with phosphate buffered saline (PBS), cells were scrapped on ice into lysis buffer (Cell Signaling Technology Inc., Danvers, MA, USA) supplemented with 1 mM PMSF and the soluble fraction was isolated after centrifugation (14000 rpm, 10 min, 4°C). Protein was quantified using a BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA) according to manufacturer’s protocol, and lysates were resolved on 8% poly-acrylamide gels and transferred onto Immobilon-P PVDF membrane (Bio-Rad, Hercules, CA, USA). Membranes were blocked in 5% milk in Tris buffered saline with 0.1% Tween-20 (TBST) for 1 hr, followed by an overnight incubation at 4°C in primary antibody (1:1000). Membranes were washed with TBST before and after exposure to goat-anti-rabbit HRP secondary antibody (1 hr; Cell Signaling) and protein were visualized using Kodak 1D Image Analysis Software 3.6 and a Kodak Image Station 2000R (Eastman Kodak Company, Rochester, NY, USA). Primary antibodies used for western blotting include phospho-p44/42 MAPK (ERK1/2), p44/42 MAPK (ERK1/2), phospho-AKT, AKT, phospho-JNK, phospho-IKK, phospho-NF-κB, phospho-elF-2α, and phospho-TAK1 were obtained from Cell Signaling, anti-GPR120 was obtained from Abcam and anti-β-actin and anti-TNFα were obtained from Sigma.
6
Antibody-based Signal Pathway Detection
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SYBR PCR Master Mix was purchased from Kapa Biosystems (Boston, MA, USA). Anti-mouse and anti-rabbit IgG-conjugated HRP and anti-mouse actin antibodies were purchased from Sigma-Aldrich (St Louis, MO, USA). Antibodies against ERK, phospho-ERK, JNK, phospho-JNK, p38, phospho-p38, Akt, phospho-Akt, p65, phospho-p65, IKK, and phospho-IKK were obtained from Cell Signaling Technology (Cambridge, MA, USA).
7
Antibody Characterization for UPR Pathway
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Antibodies against proliferating cell nuclear antigen, binding immunoglobulin protein (BIP), protein disulfide isomerase (PDI), activating transcription factor-4 (ATF4), JNK, phospho-JNK, phospho-eukaryotic translation initiation factor 2A (eIf2α), p58 inhibitor of the PKR kinase (p58IPK), poly ADP ribose polymerase (PARP), caspase-3, caspase-9, NF-κB subunits p50, p52 and p65, IκB and phospho-IKK were purchased from Cell Signaling Technology (Danvers, MA). Antibody against actin was from Sigma-Aldrich, antibody against SREBP-1c was from Millipore (Burlington, MA), and that against ATF6 was from Santa Cruz Biotechnology (Dallas, TX). Antibody against XBP1 was from Abcam and that against phospho-Ire1α from ThermoFisher Scientific.
8
Protein Quantification and Western Blot Analysis
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To enable loading of equal amounts, protein concentrations were determined with a bicinchoninic acid protein assay reagent kit (Beyotime, China). The protein samples were adjusted to the same concentration before loading. HL-7702 cell and liver tissue protein extracts were fractionated by electrophoresis on 12% SDS-polyacrylamide gels (Beyotime, China) and then transferred to nitrocellulose membranes. The protein blots were blocked with 5% BSA dissolved in Tris-buffered saline with Tween 20 and then incubated with appropriate primary antibodies against NF-κB p65 (1:500; Proteintech, 10745-1-AP), p-NF-κB p65 (1:500; Cell Signaling Technology, #3031), cyclin D1 (1:500; Cell Signaling Technology, #2978), IKK (1:1000; Proteintech, 15649-1-AP), phospho-IKK (1:500; Cell Signaling Technology, #2694), cleaved caspase-3 (1:1000; Cell Signaling Technology, #9664), GAPDH (1:1000; Proteintech, 60004-1-1g), and β-actin (1:2000; Cell Signaling Technology #8457) at 4 °C for 12 h. The membranes were then washed 3 times and incubated with a 1:3000 dilution of horseradish peroxidase-conjugated secondary antibody (Yeasen, China) for 2 h. Then, the proteins were detected with an enhanced chemiluminescence system (Bio-Rad, USA).
9
Fluorofenidone Modulates Inflammatory Responses
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Fluorofenidone (AKFPD) was synthesized by Haikou Pharma (Haikou, China). LPS (Escherichia coli O111:B4) was purchased from Sigma Aldrich (#L2630). The antibody against cleaved caspase-3 (#9664), BCL-2 (#2876), Bax (#2772), phospho-ERK (#4370), phospho-JNK (#4668), phospho-P38 (#4631), ERK (#4695), JNK (#9252), P38 (#8690), phospho-IKK (#2697), phospho-IκB (#2859), IKK (#2682), IκB (#9242), P65 (#4764), anti-mouse IgG HRP-linked antibody (#7076), and anti-rabbit IgG HRP-linked antibody (#7074) were purchased from Cell Signaling Technology. The antibody against phospho-P65 (#ab76302) was from Abcam. The antibody against α-tubulin was purchased from proteintech (#66031-1-Ig). The F4/80 antibody was from BD bioscience (#565409), and the MPO antibody was from proteintech (#22225-1-AP). The BCA Protein Assay kit was from Thermo Fisher Scientific (#23225). A terminal deoxynucleotidyl transferase–mediated nick end labeling (TUNEL) kit was purchased from Roche (#11684817910). An FITC annexin V and propidium iodide (PI) staining kit was purchased from BD Biosciences (#556547). An ELISA test kit of IL-1β (#88-7013-88), TNF-α (#88-7324-22), and IL-6 (#88-7064-22) was purchased from Thermo Fisher Scientific. All other chemicals were of analytical grade.
10
Western Blotting and IHC Antibodies for Inflammation
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Antibodies used for western bloting included F4/80 (rat monoclonal, Abcam, ab16911; 1:200), CD11b (rabbit monoclonal, Abcam, ab133357; 1:1000), LY6C (rat monoclonal, Novus, NBP2-00441; 1:1000), MCP-1 (rabbit polyclonal, Cell Signaling Technology, #2029; 1:1000), NLRP3 (rabbit monoclonal, Cell Signaling Technology, #15101; 1:1000), pro-Caspase1 (rabbit monoclonal, Abcam, ab179515; 1:1000), GSDMD (rabbit monoclonal, Abcam, ab209845; 1:1000), IKK (rabbit monoclonal, Abcam, ab178870; 1:1000), phospho-IKK (rabbit monoclonal, Cell Signaling Technology, #2697; 1:1000), p65 (mouse monoclonal, Sabta cruz, sc-8008; 1:200), phospho-p65 (rabbit monoclonal, Cell Signaling Technology, #3033; 1:1000), IĸBα (mouse monoclonal, Cell Signaling Technology, #9246; 1:1000), phospho-IĸBα (mouse monoclonal, Cell Signaling Technology, #4814; 1:1000), GAPDH (mouse monoclonal, Abcam, ab8245; 1:8000), Goat Anti-Mouse IgG (Abcam, ab97023; 1:10000), Goat Anti-Rabbit IgG (Abcam, ab97051; 1:10000) and Goat Anti-Rat IgG (Abcam, ab97057; 1:10000). Antibodies used for immunohistochemistry and immunohistochemistry-frozen included F4/80 (rat monoclonal, Abcam, ab16911; 1:50) and LY6C (rat monoclonal, Novus, NBP2-00441; 1:200). LPS, IFN-γ, IL-4, IL-10 and M-CSF were purchased from PeproTech, ATP and nigericin were purchased from Med Chem Express.
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