Western blot analysis was performed as previously described.26 (link), 27 (link) Primary antibodies included anti-phospho-p65 antibody (Cell signaling #3033, 1:1000), anti-p65 antibody (Cell Signaling #6956, 1:1000), anti-IκBα antibody (Cell Signaling #3033, 1:1000), anti-IκBα antibody (Cell Signaling #9242, 1:1000), anti-phospho- IκBα antibody (Cell Signaling #6956, 1:2859) and anti-α-tubulin (Sigma-Aldrich #T-9026, 1:5000).
Anti iκbα antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-IκBα antibody is a specific antibody that recognizes the IκBα protein. IκBα is a key regulator of the NF-κB signaling pathway, which plays a crucial role in various cellular processes. This antibody can be used to detect and monitor the expression levels of IκBα in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti akt antibody, by Cell Signaling Technology (3 mentions)
Anti erk1 2 antibody, by Cell Signaling Technology (3 mentions)
Anti phospho jnk antibody, by Cell Signaling Technology (2 mentions)
Anti nf κb p65 antibody, by Cell Signaling Technology (2 mentions)
Protein a g plus agarose, by Santa Cruz Biotechnology (2 mentions)
Anti phospho akt antibody, by Cell Signaling Technology (2 mentions)
Chemi lumi one super, by Nacalai Tesque (2 mentions)
Anti phospho p65 antibody, by Cell Signaling Technology (2 mentions)
Anti p65 antibody, by Cell Signaling Technology (2 mentions)
Anti phospho iκbα antibody, by Cell Signaling Technology (2 mentions)
Anti jnk antibody, by Cell Signaling Technology (2 mentions)
Sp600125, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Nacalai Tesque (2 mentions)
Hrp conjugated anti rabbit antibody, by ZSGB-BIO (1 mentions)
Enhanced ecl kit, by Beyotime (1 mentions)
Rabbit anti phospho p38, by Cell Signaling Technology (1 mentions)
Anti phospho erk, by Cell Signaling Technology (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Bca assay kit, by Beyotime (1 mentions)
22 protocols using anti iκbα antibody
1
Western Blot Protein Immunodetection
2
Phospho-protein Analysis of Dendritic Cells
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DCs treated with PLP-2 at 100 μg/mL or LPS at 1 μg/mL for 24 h were collected by centrifugation and washed three times with pre-cold phosphate buffer (PBS). Then total proteins of DCs were prepared using KGP950 Phosphorylated Protein Extraction kit (KeyGEN Biotech, Jiangsu Province, China). Besides, the nuclear and cytoplasmic proteins were prepared using Nuclear and Cytoplasmic Protein Extraction kit (Beyotime, China). Protein concentration was determined using BCA assay kit (Beyotime, China).
Total proteins were separated by electrophoresis using a 10% SDS polyacrylamide gel and transferred onto a nitrocellulose membrane. Membrane was blocked using Tris-buffered-saline with Tween-20 (TBST) containing 5% BSA (Solarbio) at 25 °C for 1 h, and incubated with primary rabbit anti-phospho-p38, anti-phospho-ERK or anti-phospho-JNK antibody (All from Cell Signaling Technology, USA) for 12 h at 4 °C. The membrane was then incubated with secondary horse radish peroxidase (HRP)-conjugated anti-rabbit antibody (ZSGB-BIO, Beijing, China) for 1 h at 37 °C. Finally, membrane was developed with enhanced ECL kit (Beyotime, China). Chemiluminescence detection was performed on ChemiDoc XRS+ imaging system (BIO-RAD). Similarly, IκB-α in the cytoplasma extracts were also determined by Western blot analysis with anti-IκB-α antibody (Cell Signaling Technology, USA).
Total proteins were separated by electrophoresis using a 10% SDS polyacrylamide gel and transferred onto a nitrocellulose membrane. Membrane was blocked using Tris-buffered-saline with Tween-20 (TBST) containing 5% BSA (Solarbio) at 25 °C for 1 h, and incubated with primary rabbit anti-phospho-p38, anti-phospho-ERK or anti-phospho-JNK antibody (All from Cell Signaling Technology, USA) for 12 h at 4 °C. The membrane was then incubated with secondary horse radish peroxidase (HRP)-conjugated anti-rabbit antibody (ZSGB-BIO, Beijing, China) for 1 h at 37 °C. Finally, membrane was developed with enhanced ECL kit (Beyotime, China). Chemiluminescence detection was performed on ChemiDoc XRS+ imaging system (BIO-RAD). Similarly, IκB-α in the cytoplasma extracts were also determined by Western blot analysis with anti-IκB-α antibody (Cell Signaling Technology, USA).
3
Lung Protein Extraction and Western Blot
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Extractions of proteins from the lung tissues were performed with T-PER tissues proteins extractions reagent kits (Beyotime Institute of Biotechnology). Extractions of nuclear and cytoplasmic proteins from the lungs were performed with nuclear and cytoplasmic proteins extractions reagent kits (Beyotime Institute of Biotechnology). Protein contents were measured using BCA protein assay kits and equal amounts of protein were added in per well on 10% SDS PAGE. Then, proteins were separated and transferred into PVDF membranes by an Electrophoresis System (Bio-Rad Co., Ltd., Hercules, USA). The resulting membranes were blocked with Tris-buffered-saline containing 0.05% Tween-20 (TBS-T), supplemented with 5% skimmed milk at room temperature for 2 hours and followed by TBS-T washings. Then membranes were incubated with related specific primary antibodies anti-NF-κB p65 antibody, anti-IκB α antibody (Cell Signaling Technology Co., Ltd., MA, USA), and anti-TLR4 antibody (Santa Cruz Co., Ltd., TX, USA) at 4°C overnight, respectively, followed by washes with TBS-T and incubation with the peroxidase-conjugated secondary antibody at room temperature for 1 hour. The detections of labeling proteins were performed with enhanced-chemiluminescence western-blotting detections kits. And the relative protein levels were normalized to β-actin (Santa Cruz Co., Ltd.) protein as the internal standard.
4
Immunoprecipitation of IκBα and Ubiquitin
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siNC or siTRIM27 was transfected. Cells were lysed in RIPA buffer (1% SDS) by sonication. Lysates were incubated with Protein A/G PLUS-agarose (sc-2003; Santa Cruz Biotechnology, USA) for 1 h. IgG (sc-2027; Santa Cruz Biotechnology, USA) was added to all samples and incubated overnight at 4 °C. Nuclear pellets were collected following centrifugation at 3000 rpm at 4 °C, followed by washing with Protein A/G PLUS-agarose beads four times. Proteins were loaded onto 4–20% gradient SDS-PAGE gels. Signals were detected by anti-IκBα antibody (#4812; Cell Signaling Technology, Danvers, MA, USA) and anti-ubiquitin antibody (ab7780; Abcam, UK).
5
Western Blot and Immunofluorescence Analysis of DENV
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Western blot analysis was performed as described previously [10] (link), [21] (link), using the following primary antibodies: anti-DENV antibody D1-11 (anti-DENV2 E, monoclonal) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-DENV prM antibody (GeneTex, Alton Pkwy Irvine, CA), anti-actin antibody (Sigma-Aldrich, St. Louis, MO) and anti-IκBα antibody (Cell Signaling Technology, Danvers, MA). Protein bands were revealed by horseradish peroxidase-conjugated antibody and enhanced chemiluminescence using a commercial kit (Thermo Fisher Scientific, Rockford, IL) by following the manufacturer's suggested protocols. Immunofluorescence staining was carried out using anti-DENV antibody D1-11 (anti-DENV2 E, monoclonal) (Santa Cruz Biotechnology, Santa Cruz, CA) and Rhodamine-conjugated secondary antibody (Jackson ImmunoResearch Laboratories Inc, West Grove, PA), and the images were captured using the AxioVision Rel.4.6 computerized image analysis system.
6
Western Blot Analysis of Cellular Proteins
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Cell extracts were diluted in sample buffer (50 mmol/L Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate, 10% glycerol, 6% 2-mercaptoethanol, and 0.0025% bromophenol blue) and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Samples were loaded at the same protein concentration for each experiment. The primary antibodies used were anti-LMP1 antibody (S12) at 1:50, anti-actin antibody (AC-74, Sigma, St. Louis, MO) at 1:5000, anti-phospho-AKT antibody (#4058, Cell Signaling Technology, Danvers, MA) at 1:1000, anti-AKT antibody (#9272, Cell Signaling Technology) at 1:1000, anti-NFκB (p65) antibody (610868, BD Biosciences, Franklin Lakes, NJ) at 1:250, anti-IκBα antibody (#4814, Cell Signaling Technology) at 1:1000, anti-caspase-3 antibody (#9662, Cell Signaling Technology) at 1:1000, and anti-poly(ADP-ribose) polymerase (PARP) antibody (C-2-10, Sigma) at 1:2000. The secondary antibodies used were Goat Anti-Mouse Ig's HRP Conjugate (AMI3404, BioSource International, Camarillo, CA) and HRP-Goat Anti-Rabbit IgG (H+L) (656120, Invitrogen, Carlsbad, CA). The bands were visualized using WEST-oneTM Western Blot Detection System (iNtRON Biotechnology, Seongnam, Korea) or Chemi-Lumi One Super (Nacalai tesque, Kyoto, Japan).
7
IκBα Quantification via Western Blot
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IB3–1 cells were lysed in M2 buffer (20 mM pH 7.0 Tris, 0.5% NP-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 2 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 20 mM β-glycerol phosphate, 10 mM, 4-nitrophenyl phosphate disodium salt, 1 mM sodium vanadate, 1 mg/ml of leupeptin). 20 μg of the cell lysate protein from each sample were fractionated by SDS-PAGE and immunoblotted. Blots were visualized with chemiluminescent substrate (Millipore, Rockville, MD) and band densities were measured using the NIH ImageJ program [40 (link)]. Anti-IκBα antibody was from Cell Signaling Technologies (Boston, MA). The antibody for β-actin was from Sigma (Sigma-Aldrich, St. Louis, MO). Differential statistics were calculated between groups using two-tailed t-tests after normalizing individual experimental values to TNFα-treated controls (n = 3 biological replicates). Westerns were also performed on the WES instrument (Protein Simple, San Jose, CA) according to manufacturer’s instructions.
8
Immunofluorescence and Immunohistochemistry Protocols
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Immunofluorescence was performed by fixing cells with 4% PFA, permeabilizing them with 0.5% Triton X-100 and blocking with bovine serum albumin for 30 minutes. After blocking, cells were incubated with antibody at 1:100 at room temperature for 2 hours, followed by incubation with 1:500 secondary antibodies Alexa fluor-488 (Invitrogen # A-11078) and Alexa fluor-543 (Invitrogen # A-11030) at room temperature for 1 hour. Nuclei were stained with propidium iodide or DAPI for 5 minutes. Immunohistochemistry experiments were performed on formalin-fixed, paraffin-embedded tissues using anti-IκBα antibody (Cell Signaling, #4814) according to manufacturer's protocols.
9
Andrographolide Modulates NF-κB Signaling
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Dulbecco's modified Eagle's medium (DMEM), trypsin (0.25%), L-glutamine, penicillin/streptomycin, and fetal bovine serum (FBS) were purchased from Gibco (Gaithersburg, MD, USA). Andrographolide (≥98%), TNF-α, LY294002, SP600125, and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The anti-iNOS rabbit polyclonal antibody (pAb) and the anti-p65 antibody were purchased from Santa Cruz Biotechnology (Dallas, TX, USA); the anti-α-tubulin mouse monoclonal antibody (mAb) was purchased from Thermo Scientific (Waltham, MA, USA); and the anti-phospho-p38 MAPK Thr180/Tyr182 rabbit pAb, anti-p38 MAPK, anti-phospho-p44/p42 extracellular signal-regulated kinase (ERK1/2) Thr202/Tyr204 rabbit pAb, anti-ERK1/2 antibody, anti-phospho-JNK Thr183/Tyr185 rabbit mAb, anti-JNK antibody, anti-phospho-Akt Ser473 rabbit pAb, anti-Akt antibody, anti-phospho-p65 Ser536 rabbit pAb, and anti-IκBα antibody were purchased from Cell Signaling (Danvers, MA, USA). A hybond-P polyvinylidene difluoride (PVDF) membrane, an enhanced chemiluminescence (ECL) western blotting detection reagent and analysis system, the horseradish-peroxidase- (HRP-) conjugated donkey anti-rabbit immunoglobulin G (IgG), and the sheep anti-mouse IgG were acquired from Amersham (Buckinghamshire, UK). Andrographolide was dissolved in 0.1% DMSO and stored at 4°C until it was used.
10
Elastase Inhibition and Inflammatory Pathways
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Human sputum NE was acquired from Elastin Product Company (Owensville, MO, USA). The NE was dissolved in a solution containing 50% 0.02 M NaOAc (pH 5) and 50% glycerol. LPS, phenylmethylsulfonyl fluoride (PMSF), and rosiglitazone were obtained from Sigma-Aldrich (St. Louis, MO, USA). The selective elastase inhibitor Elaspol was purchased from Ono Pharmaceutical Co., Ltd. (Osaka, Japan). The anti-IκBα antibody was obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-peroxisome proliferator-activated receptor gamma (PPARγ), anti-TLR4, anti-p65, anti-NE, and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
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