Recombinant murine sRANKL (cat. no. 315-11C) was purchased from PeproTech, Inc. The antibodies used for fluorescent microscopy were as follows: Anti-VASP (cat. no. 13472-1-AP; 1:100; ProteinTech Group, Inc.), Phalloidin (cat. no. P5282; 1:100; Sigma-Aldrich; Merck KGaA), DAPI (1:1,000; cat. no. C1002; Beyotime Institute of Biotechnology). Antibodies used for western blotting experiments were anti-GAPDH (cat. no. 60004-1-Ig; 1:50,000; ProteinTech Group, Inc.), anti-VASP (cat. no. 13472-1-AP; 1:1,000; ProteinTech Group, Inc.), anti-TRAP (cat. no. sc-376875; 1:1,000; Santa Cruz Biotechnology, Inc.), anti-αV integrin (cat. no. 10569-1-AP; 1:1,000; ProteinTech Group, Inc.), anti-IκBα (cat. no. 10268-1-AP; 1:1,000; ProteinTech Group, Inc.), anti P-IκBα (cat. no. 13921-1; 1:1,000; Cayman Chemical Company), anti-NF-kB p65 (cat. no. ABP0167), anti-lamin A (cat. no. ABP0098), anti-nuclear factor of activated T cells cytoplasmic 1 (NFATc1) (cat. no. ABP53112) (all 1:1,000; Abbkine Scientific Co., Ltd.) and c-Fos (cat. no. AF6489; 1:1,000; Beyotime Institute of Biotechnology). Cy3-labeled goat anti-mouse was used as secondary antibody (cat. no. A0521; 1:500; Beyotime Institute of Biotechnology).
Anti iκbα
Manufactured by Proteintech
Sourced in United States, China
Anti-IκBα is a laboratory reagent used to detect the IκBα protein. IκBα is an inhibitor of the NF-κB transcription factor, which plays a key role in regulating immune and inflammatory responses. This antibody can be used in various applications, such as Western blotting, immunoprecipitation, and immunohistochemistry, to study the expression and localization of IκBα in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti nf κb p65, by Proteintech (2 mentions)
Anti p65, by Proteintech (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Bca assay kit, by Beyotime (1 mentions)
Hrp conjugated secondary antibody, by Boster Bio (1 mentions)
Anti β actin, by Boster Bio (1 mentions)
Rabbit anti tlr4, by Abcam (1 mentions)
Anti histone 3, by Proteintech (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Ecl kit, by Beyotime (1 mentions)
Anti pcna, by Proteintech (1 mentions)
Anti nlrp3, by Wanlei (1 mentions)
Anti pro caspase 1, by Wanlei (1 mentions)
Anti nrf1, by Proteintech (1 mentions)
Anti asc, by Wanlei (1 mentions)
Anti caspase1 p20, by Wanlei (1 mentions)
Anti nrf2, by Proteintech (1 mentions)
Anti proliferating cell nuclear antigen, by Cell Signaling Technology (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
7 protocols using anti iκbα
1
Osteoclastogenesis Regulation by RANKL
2
Protein Quantification and Western Blot Analysis
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Total proteins were extracted from the infarct cortex and protein concentrations were determined using a BCA assay kit as instructed by the manufacturer (Beyotime Biotech, Jiangsu, China). 1/4 volume of 5x loading buffer was added and followed by a boiling water bath for 10 minutes for denaturation. Samples, containing 40 µg proteins, were loaded and separated by electrophoresis on 12% SDS-polyacrylamide gels. Subsequently, proteins were transferred onto PVDF membranes, blocked for two hours with 5% nonfat dry milk at room temperature, and incubated with rabbit anti-TLR4 (1 : 300, Abcam, Cambridge, MA, USA), anti-NF-κB p65 (1 : 2000, Proteintech, Rosemont, USA), anti-IκBα (1 : 2000, Proteintech, Rosemont, USA), anti-β-actin (1 : 200, Boster, Hubei, China), and anti-Histone 3 (1 : 2000, Proteintech, Rosemont, USA) antibodies at 4°C on a shaker overnight and then washed, followed by incubation with the corresponding HRP-conjugated secondary antibody (Boster, Hubei, China). Finally, ECL solution was added to reveal the bands and the gray value was analyzed by ImageJ software (NIH).
3
Western Blot Analysis of NRF, PCNA, and Inflammatory Markers
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Proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE ) and transferred to polyvinylidene fluoride (PVDF ) membranes. After blocking with phosphate-buffered saline with Tween 20 (PBST) containing 5% fat-free milk, PVDF membranes were coincubated with the antibodies anti-NRF1(1: 1,000, Proteintech, Wuhan, China); anti-NRF2(1: 1,000, Proteintech); anti-PCNA(1: 1,000, Proteintech); anti-β-actin (1: 1,000, Sigma-Aldrich, St. Louis, MO), anti-IκBα (1: 1,000, Proteintech, China), anti-P50 (1: 1,000, Proteintech), anti-NLRP3 (1: 1,000, Wanleibio, Shenyang, China), anti-ASC (1: 1,000, Wanleibio), anti-pro-Caspase1(1: 1,000, Wanleibio); anti-Caspase1-p20 (1: 1,000, Wanleibio) at 4°C overnight. Subsequently, PVDF membranes were incubated with the corresponding secondary antibody at 37°C for 1 h. Proteins were detected using the ECL kit (Beyotime, Beijing), and visualized using a Tanon-5200Multi (Tanon, Shanghai, China) device. Densitometry analysis was performed using the Image J software (National Institutes of Health, Bethesda, MD).
4
Western Blot Analysis of Protein Targets
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For western blot analysis, equal amounts of protein (30–80 μg) was separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA) at 100 V for 1 h at 4°C. Blots were blocked with 5% milk in Tris Buffered Saline-Tween 20 and the following primary antibodies were used: anti-cleaved-Caspase3 (dilution 1:1,000; Cell Signaling Technology, Danvers, MA, USA), anti-proliferating cell nuclear antigen (PCNA) (dilution 1:1,000; Cell Signaling Technology), anti-IκBα (dilution 1:500; Proteintech, Wuhan, China), anti-zona occludens 1 (ZO-1) (dilution 1:500; Proteintech), anti-occludin (dilution 1:500; Proteintech), anti-p-NF-κB (p-P65) (1:500; Cell Signaling Technology); anti-LaminB1 (1:500; Proteintech) and anti-GAPDH (dilution 1:5,000; Sigma, St. Louis, MO, USA).The membranes were analysed by the use of super ECL detection reagent (Applygen, Beijing, China).
5
Investigating Hippocampal Protein Regulation
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Briefly, rat hippocampus tissues and cultured cells were lysed in RIPA lysis buffer (Beyotime, China). After centrifuging at 14,000g at 4 °C for 30 min, the supernatant was collected and separated by 10% SDS-PAGE gels and transferred onto PVDF membrane (Millipore, USA). After being blocked with 5% non-fat milk, the membrane was incubated overnight at 4 °C with the following primary antibodies: anti-PDYN (1:1000; Abcam), anti-TLR4 (1:500; Abcam), anti-IκB-α (1:1000, Proteintech, USA), anti-phosphor-(p)-IκB-α (1:1000, Proteintech), anti-NF-κB p65(1:1000, Proteintech), anti-p-NF-κB p65 (1:1000, Proteintech), anti-caspase-3 (1:1000; Abcam), anti-Bax (1:1000, Abcam), anti-Bcl-2 (1:1000; Abcam), followed by horseradish peroxidase-labeled secondary antibodies (1:5000; Proteintech) for 1 h. Quantity One (Bio-Rad Laboratories, USA) software was used to analyze the results. β-actin and α-tubulin were used as the internal references.
6
Antibody Validation for m6A Studies
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The following antibodies were used in this study: anti-m6A (Synaptic Systems, 202003 for m6A-seq; Abcam, ab208577 for m6A-IP qPCR), anti-YTHDF1 (Proteintech, 66745–1-lg), anti-TRAF6 (Abcam, ab181622), anti-TRAF3 (Proteintech, 18099-1-AP), anti-β-actin (HuaAn, R1207–1), anti-Flag (Sigma-Aldrich, F3165), anti-MYC (Abcam, ab32), anti-DDX60 (Abcam, ab139807), anti-MyD88 (Proteintech, 23230-1-AP), anti-phosphor-p65 (Cell Signaling Technology, 3033), anti-phosphor-IκBα (Cell Signaling Technology, 2859), anti-TRIF (Proteintech, 23288-1-AP), anti-p65 (Proteintech, 10745-1-AP), anti-IκBα (Proteintech, 10268-1-AP), anti-BCLAF1 (Proteintech, 26809-1-AP), anti-THRAP3 (Proteintech, 19744-1-AP), anti-TRAF3 (Proteintech, 18099-1-AP), anti-phospho-IKKα/β (Cell Signaling Technology, 2078), anti-IKKα (HuaAn, ER30911), anti-IKKβ (HuaAn, ER1706-13), anti-p38 (HuaAn, ET1602-26), phospho-p38 (Cell Signaling Technology, 9211), anti-phospho-JNK (HuaAn, RT1488), anti-JNK (HuaAn, RT1550), anti-ERK1/2 (Cell Signaling Technology, 9102), phospho-ERK1/2 (HuaAn, ET1610–13), normal rabbit IgG (Cell Signaling Technology, 2729) and normal mouse IgG (Cell Signaling Technology, 5946).
7
Western Blot Immunodetection Protocol
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Western blot was performed as previously reported44 (link). Briefly, the membranes were incubated overnight at 4 °C with the following corresponding primary antibodies: anti-HECTD1 (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-HECTD2, anti-HECTD3 (1:800, BIOS, Beijing, China), anti-TRAF3 (1:200, Affinity Biosciences, Shanghai, China), anti-phospho-IKKβ (Tyr199), anti-phospho-IκBα (Ser32/Ser36), anti-phospho-p65 (Ser536) (1:500, Affinity Biosciences, Shanghai, China), anti-IKKβ, anti-IκBα, anti-p65 (1:1000, Proteintech, Wuhan, China), anti-GST tag, anti-His tag, anti-FLAG tag, anti-HA tag (1:5000, Proteintech, Wuhan, China), and anti-β-actin (1:3000, Proteintech, Wuhan, China) antibodies.
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