The total protein concentration was measured using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology, Haimen, China). In brief, 30 μg of proteins were loaded into each lane of sodium dodecyl sulfate polyacrylamide gels, separated by electrophoresis, and then transferred to polyvinylidene fluoride membranes (Pall Corporation, Beijing, China), which were incubated with primary Abs against IRAK4, phospho-IRAK4, P38, phospho-P38, JNK, phospho-JNK, TRIF, IRF3, phospho-IRF3, NF-κB p65, phospho-NF-κB p65, IKKα, IKKβ, phospho-IKKα/β, IkBα, and phospho-IkBα (Cell Signaling Technology, Inc., Beverly, MA, USA; all diluted to 1:1000), as well as GAPDH (1:5000) and PSGL-1 (Santa Cruz Biotechnology, Inc.; 1:1000). Anti-rabbit or mouse horseradish peroxidase-linked Ab (ZSGB-BIO Technology Co., Ltd., Beijing, China; 1:8000) was used as the secondary Ab. Detection was performed using a chemiluminescence kit (Advansta, Inc., Menlo Park, CA, USA). Densitometry of proteins was analyzed with Gel-Pro software (Media Cybernetics).
Phospho ikbα
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-IkBα is a laboratory reagent used to detect and measure the phosphorylation of the IkBα protein. IkBα is a regulatory protein that inhibits the activity of the NF-kB transcription factor. Phosphorylation of IkBα leads to its degradation, allowing NF-kB to translocate to the nucleus and regulate gene expression.
Automatically generated - may contain errors
Lab products found in correlation
Phospho ikkα β, by Cell Signaling Technology (2 mentions)
Phospho akt, by Cell Signaling Technology (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
α tubulin, by Cell Signaling Technology (2 mentions)
Phospho irak4, by Cell Signaling Technology (1 mentions)
Gel pro software, by Media Cybernetics (1 mentions)
Phospho irf3, by Cell Signaling Technology (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride membrane, by Pall Corporation (1 mentions)
Bicinchoninic acid assay kit, by Beyotime (1 mentions)
Chemiluminescence kit, by Advansta (1 mentions)
Phospho nf κb p65, by Cell Signaling Technology (1 mentions)
Psgl 1, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Ikkα β sc 7607, by Santa Cruz Biotechnology (1 mentions)
Iblot dry blotting system, by Thermo Fisher Scientific (1 mentions)
Ez ecl chemiluminescence detection kit, by Sartorius (1 mentions)
Jsh 23, by Selleck Chemicals (1 mentions)
Phospho pi3k, by Cell Signaling Technology (1 mentions)
Histone h3 17168 1 ap, by Proteintech (1 mentions)
10 protocols using phospho ikbα
1
Western Blot Analysis of Immune Signaling
2
Quantification of IKK and IκB Proteins
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Total protein was separated by SDS-PAGE and transferred onto PVDF membranes by the iBlot Dry Blotting System (Invitrogen, MA, USA). Blots were incubated overnight at 4 °C with primary anti-human antibodies for phospho-IKK α/β (2697 S, 1:1000; Cell Signaling Technology, MA, USA), IKK α/β (sc-7607,1:400; Santa Cruz Biotechnology, TX, USA) phospho-IKB-α (9246 S, 1:200; Cell Signaling Technology) and IKB-α (sc-203, 1:200; Santa Cruz Biotechnology). Thereafter, the blots were incubated with either donkey anti-rabbit (Biolegend, CA, USA) or anti-mouse (Cell Signaling Technology) HRP-linked antibody (1:2000). To assess housekeeping protein expression, membranes were reblotted overnight at 4 °C with primary rabbit anti-human β-actin HRP conjugate (Cell Signaling Technology). Bands were visualized using the EZ-ECL chemiluminescence detection kit (Biological Industries, Israel). Total IKK α/β, IKB-α and β-actin were used as internal controls when applicable to verify basal level expression and equal protein loading. Further details included as Supplementary Information.
3
Chondroprotective Signaling Pathways Inhibition
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Recombinant rat IL-1β (501-RL-010) was obtained from R&D systems (Minneapolis, MN, USA). Antagonist AZ3451 was obtained from MedChem Express (Monmouth Junction, NJ, USA). The p38 MAPK inhibitor SB203580, the NF-κB nuclear translocation inhibitor JSH-23 and the P13K/AKT inhibitor LY294002 were obtained from Selleck chemicals (Houston, TX, USA). The antibodies against P38(#8690), phospho-P38(#4511), ERK ½ (#4695), phospho-ERK ½ (#4370), JNK (#9258), phospho-JNK (#9255), phospho-P65(#3033), phospho-IKBα (#2859), phospho–IKKαβ (#2697), IKKβ (#8943), AKT (#4685), Phospho-AKT (#4060), Phospho–PI3K (#17366), mTOR (#2983), Phospho-mTOR (#5536), COX2(#12282), Atg5 (#12994), Atg7(#8558), Atg12(#4180), Beclin1(#3495), LC3A/B (#12741), Cleaved Caspase-3 (#9664) and p16INK4A (#80772) were purchased from Cell Signaling Technology (Danvers, MA). Antibody against Collagen Type II(15943-1-AP), MMP1 (10371-2-AP), IKBα (10268-1-AP), P65 (10745-1-AP), Histone-H3(17168-1-AP), Cytochrome C (10993-1-AP), Caspase 3 (19677-1-AP), Bax (50599-2-Ig), Bcl-2 (12789-1-AP) and PI3K (20584-1-AP) were purchased from Proteintech Group (Wuhan, China). Antibodies against iNOS (ab3523), MMP13 (ab39012), PAR2 (ab180953), SOX9 (ab185966) and Aggrecan (ab36861) were supplied by Abcam (Cambridge, UK). Antibodies against ADAMTS5 (BA3020) and GAPDH (BM3876) were obtained from Boster (Wuhan, China).
4
Protein Extraction and Western Blot Analysis from LV Tissues
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Protein from LV tissues was extracted with Radio-Immunoprecipitation Assay (RIPA) lysis buffer, and western blot analysis was performed as described previously.13 (link),16 (link),17 (link) In brief, after blocking nonspecific binding with 5% BSA, membranes were incubated overnight at 4°C with primary antibodies against collagen I (1310-01; Southern Biotech), collagen III (1330-01; Southern Biotech), α-smooth muscle actin (ab230458; Abcam), fibronectin (sc-6953; Santa Cruz Biotechnology), GAPDH (Chemicon; Merck), phospho-NF-κB/phospho-p65 (ab86299; Abcam), phospho-IkBα (#2859; Cell Signaling Technology), IkBα (sc-371; Santa Cruz Biotechnology), phospho-Smad3 (#9520; Cell Signaling Technology), Smad3 (51-1500; Invitrogen, Waltham, MA, USA), Smad7 (sc-11392; Santa Cruz Biotechnology), and Smurf2 (sc-393848; Santa Cruz Biotechnology). After being washed, the membranes were incubated with LI-COR IRDye 800-conjugated secondary antibodies, anti-mouse (#24849; Rockland Immunochemicals, Limerick, PA, USA) and anti-rabbit (#36595; Rockland Immunochemicals), in the dark for 1 h at room temperature. Signals were scanned and visualized by Odyssey Infrared Imaging System (Li-COR Biosciences, Lincoln, NE, USA). The ratio of the target protein was subjected to GAPDH and was quantified with ImageJ software (National Institutes of Health, Bethesda, MD, USA).
5
Protein Extraction and Western Blot Analysis
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Total proteins form cells and tissues were isolated using RIPA buffer (Millipore, Boston, MA, USA) supplemental with protease inhibitor cocktail (Roche, Basel, Switzerland). Nuclear proteins were isolated using nuclear protein isolation kit (KGP1100, KeyGEN BioTECH, Nanjing, China). Twenty micrograms of protein suspended in SDS loading buffer were run on 10% SDS-PAGE gels and electrotransferred to PVDF membranes (Millipore). Anti-TRIM31 (1:1,000, an67785, Abcam, Cambridge, UK), p65 (1:1,000, 8242, Cell Signaling, Danvers, CO, USA), p84 (1:1,000, ab487, Abcam), IkBα (1:1,000, 4812, Cell Signaling), phospho-IkBα (1:1,000, 2859, Cell Signaling), and α-Tubulin (1:1,000, 3873, Cell Signaling) antibodies were used.
6
Protein Extraction and Western Blotting
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Whole-cell extracts were obtained from 1.5 × 106 cells using RIPA buffer (Cell Signaling Technologies Inc., Danvers, MA). For cytoplasmic and nuclear protein extraction, the Nuclear Extract kit (Active Motif, Carlsbad, CA, USA) was used, and extraction was carried out according to the manufacturer’s instructions. Western blotting analyses were performed as previously reported [54 (link)], with primary antibodies against β-actin (Sigma-Aldrich), NF-κB (Santa Cruz, Dallas, Texas, USA), α-tubulin, PARP1, phospho-IkBα and phospho-NF-κB (Cell Signaling Technologies). Membranes were scanned and analyzed using the Odyssey® infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA) and Odyssey 3.0 imaging software. Equal loading and quality of cytosolic and nuclear proteins were determined using α-tubulin and PARP1 expression levels, respectively. The relative protein expressions were normalized using the quantified level of β-actin expression for total lysates, α-tubulin for cytosolic extracts and PARP1 for nuclei extracts, as previously reported [54 (link)]. The data presented are representative of three separate experiments with similar results.
7
Quantifying IκBα Signaling in MCF7 Cells
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Total protein was extracted from cultured MCF7 cells (TNF-α treated or untreated) using cell lysis buffer (Cell Signaling) supplemented with 1 × Halt Protease and Phosphatase Single-use Inhibitor Cocktail (Thermo Scientific) as recommended by the manufacturers. Western blots were performed as previously described49 (link). IkBα (1:1,000) rabbit polyclonal, Phospho-IkBα (1:1,000) rabbit monoclonal and Tubulin (1:1,000) rabbit monoclonal (Cell Signaling) antibodies were used for detection of the proteins.
8
Protein Extraction and Western Blot Analysis
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Total protein was harvested with RIPA buffer (Beyotime, Jiangsu, China) containing a phenylmethylsulfonyl fluoride (PMSF, Beyotime, Jiangsu, China) and phosphatase inhibitor cocktail (Cell Signaling Technology). Protein concentrations were determined using BCA assay (Pierce, Thermo Fisher Scientific). Primary antibodies recognizing CTGF, STAT1, Phospho‐STAT1, Phospho‐P65 (all 1:1000, Cell Signaling Technology), Phospho‐IKB‐α (1:500, Cell Signaling Technology), CYP19A1, PCNA (all 1:1000, Proteintech), and cleaved‐PARP (1:1000, Cell Signaling Technology) were incubated to examine the proteins. The α‐tubulin (1:2000, Proteintech) was used as an intrinsic control. The immunoreactive bands were photographed and analyzed with ChemiDoc MP Imaging System and Image Lab Software (Bio‐Rad, Hercules, CA, USA).
9
Alum and PS-HH2 Modulate DC Signaling
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Immature DCs derived from bone marrow were incubated and harvested on Day 5 for Western blot analysis. After treatment with alum (125 μg/ml), PS-HH2 (PS: 20 μg/ml, HH2: 40 μg/ml), or alum-PS-HH2 complex for 0, 5, 10, and 30 min, respectively, DCs were washed twice with Tris buffered saline (TBS) and lysed with RIPA buffer. The protein concentration was determined using the BCA assay. Equal amount of protein were separated by SDS-PAGE and transferred on polyvinylidene fluoride membranes (Millipore, Billerica, MA). After blocking with 5% non-fat milk /TBS-T (TBS containing 0.1% Tween 20), the blots were probed overnight at 4°C with antibodies to phospho-Syk, phospho-Akt, phospho-IkBα and phospho-NF-κB antibodies (all from Cell Signaling Technology, Beverly, MA), respectively. Blots were developed using a Supersignal West Pico chemiluminescent substrate kit (Pierce, Rockford, IL), following incubation with appropriate horseradish peroxidase-conjugated secondary antibody. GAPDH was detected on the same membrane and used as the loading control.
10
Integrin Activation and Signaling Protocols
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GAPDH, phospho-Ik Bα, total Ik Bα, and Rho kinase antibodies were purchased from Cell Signaling Technology. PIP5K1γ antibody was purchased from Abcam. α-smooth muscle actin antibody was purchased from Sigma. Integrin antibodies used were monoclonal anti-active integrin β1 (HUTS-4, Millipore), activating integrin β1 (TS2/16, Invitrogen). c15 was purchased from Tocris. β1-CHAMP was synthesized in our laboratory (16 (link)). Manganese was purchased from Fisher. Y-27632 was purchased from Sigma. SB 203580 and BAY 11–7082 were purchased from MedChemExpress. UNC3230 was purchased from Cayman Chemical. PI(4 (link),5 (link))P2 diC16 was purchased from Echelon Biosciences and used with carrier 1 per manufacturer instructions. Cycloheximide was purchased from Research Products International (RPI).
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