HMEC (2 × 105 cells/well) were cultured in complete medium and incubated overnight at 37 °C to allow attachment. HMEC were exposed to serum starvation for 2 h and then stimulated with LPS in the absence or presence of ARC. After incubation, endothelial cells were trypsinized, washed, fixed, and permeabilized as described in the BD Phosflow protocol (protocol III). Subsequently, cells were labeled with Abs against phospho-ERK1/2 (Tyr 202/Tyr 204) or IgG1κ isotype conjugated with Alexa Fluor® 488 and subjected to flow cytometry. NF-κB signaling was assessed by Western blotting. After starvation, HMEC were scraped and lysed with lysis buffer (60 mM de Tris/HCl, pH 6.8 + 1% SDS) in the presence of a cocktail of protease inhibitors (Sigma). After centrifugation, the lysates were electrophoresed and transferred to a nitrocellulose membrane. After blocking membranes were incubated with anti-phospho-ERK1/2 Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-IκBα (BD Biosciences) followed by HRP-conjugated secondary Ab. Each membrane was reprobed with an Ab against β-actin (BD Biosciences) and proteins bands were visualized by ECL.
Anti iκbα
Manufactured by BD
Sourced in United States
Anti-IκBα is a laboratory reagent used in the study of cellular signaling pathways. It is a specific antibody that binds to and detects the IκBα protein, which plays a crucial role in the regulation of the NF-κB transcription factor. This reagent can be used in various experimental techniques, such as Western blotting, immunoprecipitation, and immunocytochemistry, to investigate the activation and localization of the NF-κB signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Perm buffer 3, by BD (2 mentions)
Anti phospho erk1 2, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Anti phospho ikkα β ser176 180 16a6, by Cell Signaling Technology (1 mentions)
Anti sharpin 14626 1 ap, by Proteintech (1 mentions)
Anti β tubulin, by Merck Group (1 mentions)
Anti gapdh sc 365062, by Santa Cruz Biotechnology (1 mentions)
Anti α tubulin, by Thermo Fisher Scientific (1 mentions)
Anti phospho p70s6k thr389, by Cell Signaling Technology (1 mentions)
Anti v5, by Bio-Rad (1 mentions)
Anti hif 1α, by BD (1 mentions)
Anti p27, by BD (1 mentions)
Anti erk1 2 pt202 py204, by BD (1 mentions)
F ab 2 fragment goat anti human igm, by Jackson ImmunoResearch (1 mentions)
Megacd40l, by Enzo Life Sciences (1 mentions)
Fix buffer 1, by BD (1 mentions)
Anti phospho akt, by BD (1 mentions)
Anti akt, by BD (1 mentions)
Recombinant human il 12, by R&D Systems (1 mentions)
Il 27, by R&D Systems (1 mentions)
6 protocols using anti iκbα
1
Endothelial Cell Response to LPS and ARC
2
Antibody Immunoblot Detection Protocol
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The antibodies used for detection on immunoblots were anti-HOIL-1 N-ter, provided by H. Walczak (University College London, London, England, UK; Haas et al., 2009 (link)), anti-HOIL-1 C-ter provided by K. Iwai (Graduate School of Medicine, Kyoto University, Kyoto, Japan; Kirisako et al., 2006 (link)), anti-HOIP (PAB6229; Abnova), and anti-linear polyubiquitin chains 1F113F5/Y102L provided by Genentech (Matsumoto et al., 2012 (link)), anti-NEMO (sc-8330; Santa Cruz Biotechnology, Inc.; #611306; BD), anti-SHARPIN (14626–1-AP; ProteinTech), anti-IκBα (#610690; BD), anti-phospho-IKKα-β (ser176/180; 16A6; Cell Signaling Technology), anti-IKKβ (AM8109a, Abgent), anti-β-tubulin (T4026; Sigma-Aldrich), and anti-GAPDH (sc-365062; Santa Cruz Biotechnology, Inc.) antibodies. Species-specific secondary antibodies coupled to horseradish peroxidase were obtained from Vector Laboratories or Amersham-Pharmacia.
3
Western Blot Analysis of Signaling Pathways
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Cells were washed with ice-cold Phosphate-Buffered Saline (PBS) and then lysed in Triton X-100-containing lysis buffer. The composition of the lysis buffer was as follows: 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 2.5 mM EDTA, 2.5 mM EGTA, 20 mM NaF, 1 mM Na3VO4, 20 mM Sodium β-Glycerophosphate, 10 mM Sodium Pyrophosphate, 0.5% Triton X-100, Roche protease inhibitor cocktail and 0.1% β-Mercaptoethanol. Lysates were pre-cleared by centrifugation before use for Western blotting. Equal amounts of protein were loaded for Western blot analysis. All the following monoclonal antibodies used were obtained from Cell Signaling Technology: anti-phospho-p70S6K (Thr389) (#9234), anti-phospho-p38 (Thr180/Tyr182) (#9215), anti-phospho-JNK1/2 (Thr183/Tyr185) (#4668), anti-phospho-ERK1/2 (Thr202/Tyr204) (#9101), anti-phospho-IκBα (Ser32) (#2859), and anti-IκBα (#9242), except for anti-p27 (BD Biosciences, #610241), anti-HIF-1α (BD Biosciences, #610959), anti-α-tubulin (Molecular Probes, #236-10501) and anti-V5 (Serotec, #MCA1360). All the antibodies were used at dilutions of between 1:10,000 and 1:5000. The Western blots shown are representative of at least two independent experiments.
4
Activated B Cell Signaling Assay
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Frozen PBMCs were thawed the day before the assay and cultivated overnight in complete medium. PBMCs were first stained for dead cells and washed, and 5 × 105 PBMCs were distributed in a 96-well V-bottom plate. Cells were stimulated or not stimulated with complete medium containing 100 ng/ml Mega CD40L (Enzo Life Sciences), 20 µg/ml F(ab′)2 fragment goat anti–human IgM (Jackson ImmunoResearch Laboratories, Inc.), or 40 ng/ml PMA. Optimal stimulation time points for each readout were selected based on prior kinetic experiments performed on control PBMCs. After 5 (p-ERK1/2 measurement)- or 35 (p-P65 and IκBα measurement)-min stimulation, cells were fixed by adding Fix Buffer I (1:1 volume; BD) and incubated 10 min at 37°C. Cells were then permeabilized for 20 min at room temperatures using Perm Buffer III (BD) and stained for 3 h at room temperature with anti-CD20 (H1; BD) and IgG2b isotypic control, anti–NF-κB P65-(pS259) (BD), anti-ERK1/2 pT202/pY204 (BD), or anti-IκBα (BD). Cells were subsequently acquired on a FACS Gallios flow cytometer and analyzed with FlowJo software (v10).
5
Cytokine Signaling Pathway Modulation
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Recombinant human IL-12, IL-23, IL-27, TNF-α, and IFN-γ were purchased from R&D Systems (MN, USA). GAPDH antibodies were from Cell Signaling Technology (MA, USA). Mouse anti-TCCR/WXS-1 and anti-gp130 mAb were purchased from R&D Systems (MN, USA). Rabbit anti-IL-27 mAb was purchased from Abcam (HKSP, HK). Mouse anti-phospho-p38 mitogen-activated protein kinase (MAPK), anti-phospho-inhibitor(I)κB-α, anti-phospho-Akt, anti-p38MAPK, anti-IκB-α, and anti-Akt mAb were purchased from BD Biosciences (CA, USA). Janus kinase (JAK) inhibitor AG490, IκB-α phosphorylation inhibitor BAY1167082, phosphatidylinositol 3-OH kinase (PI3K) inhibitor LY294002, p38MAPK inhibitor SB203580, c-Jun N-terminal kinase (JNK) inhibitor SP600125, and extracellular signal-regulated kinase (ERK) inhibitor U0126 were purchased from Calbiochem Corp. (San Diego, CA, USA). In this program, the concentration of DMSO was 0.1% (vol/vol) for all data subsets.
6
Macrophage Signaling Pathway Analysis
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The addition of P3C and Mtb was staggered to enable fixation with 2% PFA to occur at the same time (time 0). Cells were fixed for 10 min with 4% PFA before the addition of an equal volume of PBS with 2 mM EDTA for detachment. Cells were vigorously pipetted to detach and then remained in fixative reagents for an hour. Macrophages were pelleted by centrifugation before resuspension in permeabilization buffer. Signaling samples were permeabilized using ice-cold Perm Buffer III (BD) for 36 h at −20C, blocked using Fc receptor specific antibody, and stained for 1h at RT with anti-IκBα (BD), anti-p-Erk1/2 (BD), anti-p-p38 (BD) and anti-pJNK (CST). Data were collected on a BD Fortessa, analyzed in FlowJo.
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