Total protein was extracted from BMMs and C3H10 cells and from the bones of mice used in the OVX experiment using a radioimmunoprecipitation assay lysis buffer (RIPA) (Sigma-Aldrich), and the protein concentration was measured using a bicinchoninic acid protein (BCA) assay kit (EMD Millipore, Bedford, MA, USA). Protein equivalents were separated using 10% SDS PAGE and transferred onto PVDF membranes (EMD Millipore). The membranes were blocked for an hour at room temperature with 5% skim milk in Tris-buffered saline with Tween 20. After incubating primary antibodies overnight at 4 °C, the membranes were nurtured with corresponding HRP conjugated secondary antibodies (Proteintech Group, Chicago, IL, USA.) The bands were visualized employing ECL Luminous Liquid (EMD Millipore). ImageJ software was used to compute the relative grey level of proteins (NIH). Anti-TRAIL was procured from Santa Cruz Bio (Santa Cruz, CA, USA). Anti-CTSK, TRAP, NFATc1, OPG, RUNX2, OCN, RANKL, COL1a, p-P65, P65, p-P38, P38, p-JNK, JNK, p-ERK, ERK, p-IKBα, IKBα, IKKα, IKKβ, and p-IKKα/β were all purchased from Cell Signaling Technology (Danvers, MA, USA).
P ikbα
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
P-IKBα is a phospho-specific antibody that recognizes the phosphorylated form of IκBα (Inhibitor of NF-κB). IκBα is a key regulator of the NF-κB signaling pathway, which plays a critical role in immune and inflammatory responses.
Automatically generated - may contain errors
Lab products found in correlation
P ikkα β, by Cell Signaling Technology (9 mentions)
P jnk, by Cell Signaling Technology (8 mentions)
Gapdh, by Cell Signaling Technology (8 mentions)
P erk, by Cell Signaling Technology (5 mentions)
P akt, by Cell Signaling Technology (5 mentions)
P nf κb p65, by Cell Signaling Technology (4 mentions)
β actin, by Proteintech (4 mentions)
Vimentin, by Cell Signaling Technology (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Nf κb p65, by Cell Signaling Technology (3 mentions)
P ikk, by Cell Signaling Technology (3 mentions)
Gapdh, by Proteintech (3 mentions)
P nf κb, by Cell Signaling Technology (3 mentions)
Cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Nfatc1, by Cell Signaling Technology (2 mentions)
Nf κb p65, by Abcam (2 mentions)
Mmp13, by Abcam (2 mentions)
Adamts5, by Abcam (2 mentions)
P pi3k, by Cell Signaling Technology (2 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions)
42 protocols using p ikbα
1
Protein Extraction and Western Blot Analysis
2
Immunoblotting Procedure for Apoptosis, Autophagy, and Inflammation Signaling
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After pharmacological treatments, total proteins were extracted from primary cultured cells using RIPA buffer (Santa Cruz Biotechnology, CA, USA). From these extracts, 50 μg were separated by SDS polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride (PVDF) membrane (GE Healthcare, USA) in a semidry chamber Trans Blot Turbo (Bio-Rad) at 25 V 1 mA during 30 min. After blocking with 5% non-fat dry milk for 2 h, the membrane was incubated with the specific antibody overnight at 4°C on a rocking platform, washed, and then incubated with the corresponding secondary antibody for 2h at room temperature. The bolt was visualized using the SuperSignal WestFemto chemiluminescencent substrate (Pierce) in the C-DigitTM scanner (LI-COR Inc. USA).
The following antibodies used for apoptosis analysis were from Santa Cruz Biotechnology (Santa Cruz, CA): anti-PARP-1 (sc-8007), anti-caspase-3 (sc-H2777); for autophagy anti-LC3 (#2775) from Cell Signaling Technology, anti-mTOR (#AHO1232) and anti-p-mTOR (pS3448) from Invitrogen, USA; finally, regulation of inflammation was evaluated by NFKB (#8242), p-NFKB (#3033), IKBα (#4814p) and p-IKBα (#2859p) from Cell Signaling. Anti-mouse (sc-2371), anti-goat (sc-2020) and anti-rabbit (sc-2370) secondary antibodies were also purchased from Santa Cruz Biotechnology.
The following antibodies used for apoptosis analysis were from Santa Cruz Biotechnology (Santa Cruz, CA): anti-PARP-1 (sc-8007), anti-caspase-3 (sc-H2777); for autophagy anti-LC3 (#2775) from Cell Signaling Technology, anti-mTOR (#AHO1232) and anti-p-mTOR (pS3448) from Invitrogen, USA; finally, regulation of inflammation was evaluated by NFKB (#8242), p-NFKB (#3033), IKBα (#4814p) and p-IKBα (#2859p) from Cell Signaling. Anti-mouse (sc-2371), anti-goat (sc-2020) and anti-rabbit (sc-2370) secondary antibodies were also purchased from Santa Cruz Biotechnology.
3
Cell Lysis and Immunoblotting Protocol
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Cell lysis was performed with RLN-T buffer (RLN buffer plus 0.5% Triton X-100 and the complete protease inhibitors cocktail EDTA-free; Roche) at 20 × 106 cells/ml for 5 min on ice. Extracts were centrifuged at 11,000 × g for 15 min at 4°C. The primary antibodies used were as follows: anti-IF1 (1:200) (22 (link)), e-cadherin (1:250, BD Biosciences), β1 integrin (1:2, kindly provided by Carlos Cabañas, CBMSO), β-catenin (1:500, BD Biosciences), vimentin (1:1,000, Cell Signaling), NF-κB p65 (1:1,000, Abcam), pIKBα (1:500, Cell Signaling), IKBα (1:1,000, Cell Signaling), α-tubulin (1:3,000, Sigma-Aldrich), β-F1-ATPase (1:25,000) and Hsp60 (1:2,000) from Ref. (35 (link)), SDH-B (1:500, Invitrogen),α-F1-ATPase (1:1,000, Molecular Probes), Core 2 of complex III (1:500, Abcam), MTCO2 (1:500, Abcam), VDAC (1:500, Abcam), and PYGM (1:1,000, Abcam).
4
Protein Extraction and Western Blotting
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Total protein was extracted using RIPA Lysis Buffer (Beyotime, Shanghai, China) containing 1% protease inhibitor and phosphatase inhibitor (Bimake, TX, USA). Equal amounts of protein samples were subjected to 10% or 15% SDS-PAGE (Solarbio, Beijing, China), separated by electrophoresis, and transferred to nitrocellulose filter membranes. The membranes were blocked with 5% skim milk powder for 1 h at room temperature, and then, the target membranes were incubated at 4°C overnight with primary antibodies against COL2A1, ACAN, ADAMTS4, ADAMTS5, MMP3, MMP13, p16 or p21 (1 : 1000, Abcam), ADAMTS5 (1: 250, Abcam), MMP9, iNOS, COX2, NF-κB p65, p-NF-κB p65, AKT, p-AKT, p-PI3K, PI3K, p-IKBα, IKBα, p-IKK or IKK (1 : 1000, Cell Signaling Technology), or GAPDH (1 : 5000, Proteintech). After incubated with HRP-conjugated secondary antibody for 1 h at room temperature, the signal was developed using an ECL chemiluminescence detection kit (Beyotime, Shanghai, China), and images were captured on ImageQuant Las4000mini (GE Healthcare, Tokyo, Japan).
5
Western Blot Antibody Analysis Protocol
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Western blot analysis was performed as described previously [9 (link)]. The antibody information used in the experiment is shown below: p38 (#8690S, 1:1000; Cell Signaling Technology, Danvers, MA, USA), Bcl-2 (AF6139, 1:1000; Affinity Bioscience), Bax (#2772, 1:1000; Cell Signaling Technology), AMPKα (#5823, 1:1000; Cell Signaling Technology), p-AMPKα (#11818, 1:1000; Cell Signaling Technology), SIRT1 (#2493, 1:1000; Cell Signaling Technology), PGC1α (AF5395, 1:1000; Affinity), TLR4 (AF7017, 1:1000; Affinity), p65 (#8242, 1:1000; Cell Signaling Technology), p-p65 (#3033, 1:1000; Cell Signaling Technology), p-IKBα (#2859, 1:1000; Cell Signaling Technology), β-actin (AC038, 1:1000; ABclonal, Woburn, MA, USA), and horseradish peroxidase (A0208, 1:1000; Beyotime, Shanghai, China). Differences in protein transfer efficiency between blots were normalized based on the quantification of β-actin.
6
Western Blot Analysis of Signaling Proteins
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Following transfection or celastrol treatment, MDA-MB-468 and MDA-MB-231 cells were harvested and lysed in RIPA containing phenylmethanesulfonyl fluoride (Beyotime). After quantification using a BCA kit (Beyotime), protein was denatured by boiling water bath for 5 min. Then, we separated 20 μg protein samples via SDS-PAGE, transferred them to nitrocellulose membranes (Bio-Rad), and blocked them in 5% skim milk for 1 h. Subsequently, the membranes were interacted with rabbit primary antibodies overnight at 4°C, including IL-6 (#12153, Cell Signaling Technology, Danvers, MA, USA), p-IKBα (#2859), IKBα (#4812), p65 (#8242), GAPDH (#2118), and Lamin B (#13435), and then interacted with anti-rabbit horseradish peroxidase-conjugated IgG (#5127) for 2 h. The protein signaling was visualized through ECL substrate (Beyotime) and quantitated via Image Lab software (Bio-Rad). Relative protein expression was analyzed with GAPDH or Lamin B as the internal control and the normalized control, respectively.
7
Protein Extraction and Western Blot Analysis
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Cellular protein was extracted using RIPA Lysis Buffer (25 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA and 1% NP-40, Servicebio, Wuhan, China) and protease inhibitor cocktail, and the protein concentration was detected using BCA (Servicebio, Wuhan, China). Loading buffer (Servicebio, Wuhan, China) was added before boiling for 10 min for use. For electrophoresis, 10% SDS-PAGE gel was used transferred to a polyvinylidene difluoride (PVDF) membrane, and blocked with 5% skimmed milk powder. Primary antibodies used for incubation were PKCθ (13643, Cell Signaling Technology, Danvers, MA, USA), IKKα (2682, Cell Signaling Technology, Danvers, MA, USA), p-IKKα/β (2697, Cell Signaling Technology, Danvers, MA, USA), IKBα (10268-1-AP, Proteintech, Wuhan, China), p-IKBα (2859, Cell Signaling Technology, Danvers, MA, USA), P65 (ab7970, ABCAm, Cambridge, UK), p-P65 (3033, Cell Signaling Technology, Danvers, MA, USA), IL-1β (A16288, Abclonal, Wuhan, China), and β-Tubulin (10094-1-AP, Proteintech, Wuhan, China). After washing off the primary antibody with PBST, the HRP-conjugated anti-rabbit or mouse antibody (Proteintech, Wuhan, China) was incubated for 2 h at room temperature. After adding ECL Chemiluminescent kit (NCM Biotech, Suzhou, China), the target proteins were visualized using the GeneGnome XRQ system (Syngene, Cambridge, UK).
8
TLR4-NF-κB Signaling Pathway Analysis
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Tissues and cells were homogenized in 10% (wt/vol) hypotonic buffer (25 mM Tris-HCl, pH 8.0, 1 mM ethylenediaminetetraacetic acid, 5 μg/mL leupeptin, 1 mM Pefabloc SC, 50 μg/mL aprotinin, 5 μg/mL soybean trypsin inhibitor, 4 mM benzamidine) to yield a homogenate. Then, the final supernatants were obtained by centrifugation at 12,000 rpm for 20 min. Protein concentration was determined using BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) with bovine serum albumin as a standard. Then, the same amount of total protein was subjected to 10% or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by immunoblotting using the following antibodies (1:1,000): rabbit anti-TLR4, p-IkBα, p-IKKα, p-NF-κB, NF-κB, SOD1/2, XO, Nrf2, Keap1, HO-1, GAPDH (Cell Signaling Technology, Inc., Massachusetts, MA, USA), and NQO1 (Abcam). Western blot bands were observed using GE Healthcare ECL Western Blotting Analysis System and exposed to X-ray film of Kodak. Every protein expression level would be defined as the gray value (Version 1.4.2b, Mac OS X, ImageJ; National Institutes of Health, Bethesda, MD, USA) and standardized to housekeeping genes (GAPDH) and expressed as a fold of control.
9
Quantitative Western Blotting Analysis
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Western blotting analysis was performed as previously reported62 (link). An equal amount (40–80 μg) of protein samples were detected. Primary antibodies against p-MLKL (Rabbit, 1:500, ab187091; Abcam, UK), MLKL (Rabbit 1:1000, ab194699; Abcam, UK), p-RIP1 (Rabbit, 1:500, 65746; Cell Signaling Technology, USA), RIP1 (Mouse, 1:500, ab72139; Abcam, UK), caspase 8 (Rabbit, 1:1000, A0215; ABclonal, China), caspase 3 (Rabbit, 1:1000, A11319; ABclonal, China), TNF-α (Rabbit, 1:500, ab6671; Abcam, UK), IL-6 (Mouse, 1:500, ab9324; Abcam, UK), iNOS (Rabbit, 1:500, ab15323; Abcam, UK), IL-1β (Rabbit, 1:500, ab2105; Abcam, UK), NF-kB p65 (Rabbit, 1:1000; ab16502, Abcam, UK), IkBα (Mouse, 1:1000, 4814; Cell Signaling Technology, USA), p-IkBα (Rabbit, 1:1000, 2859; Cell Signaling Technology, USA); H3 (Rabbit, 1:1000, AF0009; Beyotime, China) and β-actin (Rabbit, 1:3000, AC026; ABclonal, China) were used. Protein expression levels were analyzed using ImageJ software and normalized to β-actin (National Institutes of Health, Bethesda, MD, USA). Phosphorylated protein expression was evaluated compared to total protein expression.
10
Immunoblotting Protocol for Protein Analysis
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Immunoblotting was performed as described previously57 (link). Briefly, whole-cell lysates were prepared using RIPA buffer (Sigma-Aldrich) containing protease inhibitor cocktail (Roche). Proteins were separated by running on 10% SDS-PAGE gels and transferred to PVDF membranes. After blocking with 5% skim milk in TBST, membranes were probed with appropriate primary antibodies overnight and further incubated with goat anti-rabbit IgG-HRP (Proteintech). Blots were developed using Western chemiluminescent HRP substrate (Millipore) and visualized using an imaging system (Tanon, 5200). The primary antibodies used in immunoblotting are as follows: β-actin (1:2000, Proteintech, 20536-1-AP), hnRNP F (1:1000, Thermo, PA522341), hnRNPA1 (1:1000, Cell Signaling Technology, #8443), hnRNPA2B1 (1:1000, ABclonal, A1162), p-IKKα/IKKβ (1:1000, Cell Signaling Technology, #2078), IKKβ (1:1000, Cell Signaling Technology, #8943), p-IKBα (1:1000, Cell Signaling Technology, #2859) and IKBα (1:1000, Cell Signaling Technology, #9242).
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