Western blotting was performed as described previously.19 , 20 , 21 We used the proteins from the renal cortex for Western blotting analysis. The catalogue number of antibodies used for Western blot were as following: rabbit anti‐MIF (Santa Cruz sc‐20121), goat anti‐CD74 (Santa Cruz, sc‐5438), rabbit anti‐phosphorylated NF‐κB p65 (Cell Signaling, No. 3031), mouse anti‐NF‐κB p65 (Cell Signaling, No. 6965), rabbit anti‐phosphorylated IkBα (Cell Signaling, 2859), anti‐IKBα (Cell Signaling, 9242L), rabbit anti‐TLR4 (Santa Cruz, sc‐10741) and mouse anti‐β‐actin (Santa Cruz, sc‐69879).
Anti ikbα
Manufactured by Cell Signaling Technology
Sourced in China, United States, United Kingdom
Anti-IkBα is a laboratory reagent that detects the IkBα protein, which plays a key role in the regulation of the NF-kB signaling pathway. This antibody can be used to measure IkBα levels in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Merck Group (4 mentions)
Anti phospho ikbα, by Cell Signaling Technology (3 mentions)
Anti p65, by Cell Signaling Technology (2 mentions)
Anti stat3, by Cell Signaling Technology (2 mentions)
Anti stat1, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Rabbit anti tlr4, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti mif, by Santa Cruz Biotechnology (1 mentions)
Goat anti cd74, by Santa Cruz Biotechnology (1 mentions)
Mouse anti β actin, by Santa Cruz Biotechnology (1 mentions)
Mouse anti nf κb p65, by Cell Signaling Technology (1 mentions)
Anti vinculin, by Thermo Fisher Scientific (1 mentions)
Trans blot turbo blotting system, by Bio-Rad (1 mentions)
M per lysis buffer, by Thermo Fisher Scientific (1 mentions)
Bolt 10 bis tris plus gel, by Thermo Fisher Scientific (1 mentions)
Tween 20, by Merck Group (1 mentions)
Non fat dry milk, by Bio-Rad (1 mentions)
Ab76302, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
14 protocols using anti ikbα
1
Western Blotting Protein Analysis
2
Western Blot Analysis of Caki-2 Proteins
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Caki-2 proteins were isolated by direct lysis with M-PER lysis buffer (Life Technologies). The protein content was quantified using the BCA protein assay kit (Thermo Fisher Scientific). An equal amount of protein from each sample was separated on Bolt™ 10% Bis-Tris Plus Gels (Life Technologies) and transferred to polyvinylidene fluoride membranes through Trans-Blot® Turbo™ blotting system (Bio-Rad). The membranes were blocked for one and a half h in 5% non-fat dry milk (Bio-Rad) diluted in PBS with 0.1% Tween 20 (Sigma-Aldrich) at room temperature and incubated with primary antibody overnight at 4 °C. After washing, the membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody. The following primary antibodies were used: anti-IKB-α (1:1000) (Cell Signalling Technology), anti-Vinculin (1:1000) (Thermo Fisher Scientific).
3
Western Blot Analysis of Collagen and Signaling Pathways
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The proteins were separated by SDS/PAGE with 10% polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were incubated with primary antibodies at 4 °C overnight after being blocked with 0.5% non‐fat milk powder for 1 h. Primary antibodies were as: anti‐type II collagen (ab34712; dilution 1 : 5000; Abcam, Cambridge, UK), anti‐phospho‐PI3K (ab182651; dilution 1 : 1000; Abcam), anti‐phospho‐NF‐κB (ab76302; dilution 1 : 10 000; Abcam), anti‐NF‐κB (ab16502; dilution 1 : 2000; Abcam), anti‐PI3K (cst5405; dilution 1 : 1000; Cell Signaling Technology, Boston, MA, USA), anti‐phospho‐AKT (cst4060S; dilution 1 : 2000; Cell Signaling Technology), anti‐AKT (cst4691S; dilution 1 : 1000; Cell Signaling Technology), anti‐phospho‐IKBα (cst9246; dilution 1 : 1000; Cell Signaling Technology), anti‐IKBα (cst4812; dilution 1 : 1000; Cell Signaling Technology) and anti‐GAPDH (abs83030; dilution 1 : 5000; Absin Bioscience Inc, Shanghai, China). After incubation with anti‐rabbit or anti‐mouse secondary antibodies at room temperature, the immune complexes were detected on a shaker. Membrane imaging was performed using an Alpha FluroChem Q imaging analysis system (Alpha Innotech, San Leandro, CA, USA). GAPDH was used as a control.
4
Western Blot Analysis of Osteogenic Markers
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Total proteins were extracted with lysis buffer (10 mM Tris-HCL, 1 mM EDTA, 1% sodium dodecyl sulfate, 1% Nonidet P-40, 1:100 proteinase inhibitor cocktail, 50 mM b-glycerophosphate, and 50 mM sodium fluoride). Aliquots of 20–60 mg per sample were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to the polyvinylidene fluoride membranes and blocked with 5% nonfat milk powder in PBST (PBS with 0.1% Tween). Next, they were incubated with the following primary antibodies overnight: anti-Osterix, anti-Runx2, anti-ALP, anti-P65, anti-p-P65, anti-IKBα, anti-IKK, anti-p-IKK (Cell Signaling Technology). The membranes were then incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibodies (Boster, Wuhan, China). The blots were visualized using an enhanced chemiluminescence kit (Amersham Biosciences, Piscataway, NJ, USA) according to the manufacturer’s recommended instructions.
5
Western Blot Analysis of NF-κB Pathway
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Following treatment, Leydig and TM3 cells were lysed in 1X loading buffer (5X loading buffer: 250 mM Tris-HCl pH 6.8, 10% SDS, 10% bromphenol blue, 50% glycerin and 5% β-5 Mercaptoethanol) for total protein collection. Protein concentrations were determined using the bicinchoninic acid method. The buffer, including all the extracted proteins, was then heated at 100°C for 30 min. Protein samples (50 µg/lane) were subsequently separated using SDS-PAGE on 11% gels and transferred onto nitrocellulose membranes. Upon blocking with 7% BSA dissolve in PBS at room temperature for 1 h, the membranes were incubated with primary antibodies (1:1,000) at 4°C for 12 h and then with IRDye® 800CW-conjugated donkey anti-rabbit secondary antibodies (cat. no. 925-32213; 1:5,000; LI-COR Biosciences, Lincoln, NE, USA). β-actin (1:5,000) was used as a loading control. Finally, the membranes were scanned using an Odyssey imaging system (LI-COR Biosciences). Image-Pro Plus software (version 6.0; Media Cybernetics, Inc., Rockville, MD, USA) was used for the densitometric analysis. The following antibodies were employed: Anti-IkBε (cat. no. sc-7155; Santa Cruz Biotechnology, Inc.), anti-IkBα (cat. no. 4812), anti-IkBβ (cat. no. 94101; both Cell Signaling Technology, Inc., Danvers, MA, USA), anti-β-actin (cat. no. a2228; Sigma-Aldrich; Merck KGaA) and anti-p-p65 (cat. no. ab16502; Abcam, Cambridge, UK).
6
Protein Expression Analysis by Western Blotting
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Western blotting analysis was performed as described previously [15 (link)]. Briefly, cells treated with CT for 48 h were lysed in RIPA buffer (Beyotime, Beijing, China). The protein concentrations were quantitated with Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Frederick, MD, USA). Total protein 30 µg/lane were loaded onto SDS–polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes (Amersham Bioscience, Piscataway, NJ). The membranes were blocked and incubated with (1:1000) rabbit anti-p-STAT3 (Cell Signaling, Cat: 9145L, Tyr 705), anti-STAT3 (Cell Signaling, Cat: 4904S), anti-p-JAK2 (Cell Signaling, Cat: 8082), anti-JAK2 (Cell Signaling, Cat: 3230), anti-I-kBα (Cell Signaling, Cat: 9242), anti-GAPDH (Cell Signaling, Cat: 2118) overnight at 4 °C and (1:2000) horseradish peroxidase-conjugated secondary antibody (Cell Signaling, Cat: 70,741) for 1 h at room temperature. The protein bands were visualized using the G-BOX Chemi system (Syngene, Frederick, MD).
7
Quantitative Analysis of Protein Signaling Pathways
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Cytosolic and nuclear protein extracts were obtained using the Nuclear Extract Kit (#40410, Active Motif, Active Motif, Carlsbad, CA, USA) following the manufacturer’s instructions. Lysates were resolved by SDS‐PAGE and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The following monoclonal antibodies were used: anti-p(Ser 473)Akt (Millipore); anti-Akt (Millipore); anti-IkBα (Cell Signaling Technology, #4812) anti-FOXO3A (Cell Signaling Technology, #12829S). Antibodies anti-TUBULIN (#sc-5274, Santa Cruz Biotechnology, Dallas, TX, USA) and anti-TATA-box binding protein (TBP, #sc-56795, Santa Cruz Biotechnology) were used as control of equal protein loading for cytosolic and nuclear fractions. Secondary peroxidase-conjugated antibodies (#1662408EDU, Bio-Rad Laboratories) were used. Blot images were acquired with a ChemiDocTM Touch Imaging System device (Bio-Rad Laboratories). Original blots are presented in Supplementary Figs. 9 and 10 . The ImageJ software (NIH, Bethesda, MD, USA) was used to perform densitometric analysis of western blot band intensity. For graph representation, the band intensity of the proteins of interest was normalized on the correspondent housekeeping proteins. NF-kB activity was evaluated by the TransAM Flexi NF-kB Family assay kit (#43298, Active Motif).
8
Cytokine-Mediated Chondrocyte Regulation
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Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12) Media is obtained from Hyclone (Utah, USA). Penicillin, streptomycin and fetal bovine serum (FBS) were obtained from Gibco BRL (Grand Island, NY, USA). Recombinant human and mouse IL-1β were obtained from R&D Systems (Minneapolis, MN). The antibodies used in this study are as follow: anti-sirt6 from Abcam (Cambridge, MA); anti-NF-κB p65 and anti-IKb-α from Cell Signaling Technology (Danvers, MA); anti-actin from Sigma; anti-collogan II from Chemicon (Temecula, CA); anti-MMP-13 from Santa Cruz Biotechnology (Santa Cruz, CA); Alexa-Fluor-488- and Alexa-Fluor-545-tagged second antibodies were from Molecular Probes (Eugene, OR); secondary antibodies goat anti-rabbit IRDye 800CW and goat anti-mouse IRDye 680 were from LI-COR Biosciences (Lincoln, NE).
9
Immune Receptor Signaling Pathway Analysis
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ssRNA40, R848, and other PRR ligands were from InvivoGen, phytohemagglutinin from Rembrel. Anti‐FLAG, anti‐EEA1, anti‐Rab7, anti‐SHP1, anti‐RRAGA, anti‐STAT1, anti‐Phospho‐STAT1, anti‐IkBα, anti‐pp38, anti‐phospho‐Erk were from Cell Signaling; anti‐LAMP1, anti‐beta‐tubulin, anti‐beta‐actin were from Abcam; anti‐phosphotyrosine (PY20) was from Zymed, anti‐HIV‐1 p18 (4C9) was from NIBSC. Anti‐CD4 and anti‐CD3 were from BD biosciences, anti‐HIV‐1 core antigen (KC57) was from Beckman Coulter, Violet LIVE/DEAD Cell Stain was from Invitrogen. GABARAP was from Abnova, PSMB6 and SNX5 were from Santa Cruz, BLOC1S1 was from Proteintech, Snapin was from Synaptic Systems. Anti‐rabbit Alexa Fluor 555, anti‐mouse Alexa Fluor 647, and streptavidin‐coated Dynabeads were from Invitrogen, pHrodo from Thermo Fisher, anti‐mouse HRP and anti‐goat HRP were from Sigma.
10
Colon Cancer Therapeutic Evaluation
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Crocin and doxorubicin were purchased from Analab Ltd. (Lisburn, UK). Tumour necrosis factor-α (TNF-α) was obtained from Sigma-Aldrich (St. Louis, MO, USA). All antibodies, including anti-Bcl2, anti-caspases, anti-Bax, anti-Beta actin, anti-CD31, anti-Ki67, anti-IKBα, p-IKBα, anti-NF-κB, and anti-VEGF were obtained from Cell Signalling Technology (CST) (London, UK). Human colon cancer lines (HT-29 and Caco-2 and normal HCEC cells) were purchased from American type culture collection and deposited to the internal cell bank at Ulster University. All other chemicals and reagents used were of analytical standard grade. A cell Titer 96® non-radioactive cell proliferation MTS assay single solution was purchased from Promega Corporation (Madison, WI, USA). Rosewell Park Memorial Institute (RPMI)-1640 (GIBCO, Waltham, MA, USA) medium, Trypsin-EDTA solution, Foetal calf serum, (GFR-Matrigel) (BD Biosciences, Erembodegem, Belgium), NF-κB p65 ELISA kit (CST), Transwell BD-Matrigel basement membrane matrix inserts (BD-Biosciences, Belgium), bovine type II collagen (Chondrex, Redmond, WA, USA), penicillin/streptomycin 100 units, dimethyl sulfoxide (DMSO), HEPES buffer, propidium iodide (Sigma-Aldrich, Gillingham, UK), calcium chloride, Actinomycin D, sodium bicarbonate, sodium chloride and disodium hydrogen phosphate were purchased from Merck, Kenilworth, NJ, USA.
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