After 40h incubation with indicated stimuli, mDCs were washed with ice-cold PBS and lysed using NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Scientific, 78833). Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific, 78442) was used according to the manufacturer’s instructions. Protein concentrations of cytoplasmic and nuclear extracts were measured using Pierce BCA Protein Assay Kit (Thermo Scientific, 23227). Nuclear proteins of mDCs (25 μg/group) were pre-cleared by incubation with Protein A/G Magnetic Beads (Thermo Scientific, 88802) for 1h with continuous rotation at 4°C. After removal of beads, nuclear proteins were incubated with anti-p105/p50 (Cell Signaling Technology, 13586S) or anti-RelB (Abcam, ab33917) and Protein A/G Magnetic Beads overnight with continuous rotation at 4°C. Beads were then harvested, followed by extensive wash. Immunoprecipitants were eluted and denatured by heating with SDS-Sample Buffer (Boston BioProducts, BP-111R) at 90°C for 5 minutes. Proteins were then separated by electrophoresis in SDS/PAGE and transferred to PVDF membrane (Bio-Rad, 1620177) for immunoblotting.
Anti relb
Manufactured by Abcam
Sourced in United States
Anti-RelB is a primary antibody that recognizes the RelB protein. RelB is a member of the NF-κB transcription factor family and plays a role in the regulation of gene expression.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Anti p105 p50, by Cell Signaling Technology (1 mentions)
Protein a g magnetic beads, by Abcam (1 mentions)
Sds sample buffer, by Boston BioProducts (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Ne per nuclear and cytoplasmic extraction reagents kit, by Thermo Fisher Scientific (1 mentions)
Protein a g magnetic bead, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Ab180127, by Abcam (1 mentions)
Anti lamin b, by Proteintech (1 mentions)
Anti gapdh hc301, by Transgene (1 mentions)
Epoch biotek spectrophotometer, by Agilent Technologies (1 mentions)
Quantipro bca assay kit, by Merck Group (1 mentions)
Anti stat1, by Cell Signaling Technology (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Anti irf3, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti phospho irf3, by Cell Signaling Technology (1 mentions)
4 protocols using anti relb
1
Immunoprecipitation of Nuclear Proteins
2
NLRP12 Signaling Pathway Analysis in Bone Marrow Cells
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The mEB8 and mouse bone marrow progenitor cells were lysed with RIPA buffer after twice washing with PBS. Then the proteins were quantified by BCA Protein Assay Kit. Primary antibodies used in western blotting were anti-NLRP12 (SAB3500555; Sigma-Aldrich), anti-NIK (AB191592; Abcam), anti-Relb (AB180127; Abcam), anti-ERK1/2 (9102; CST), anti-phospho-ERK1/2 (9101; CST), anti-p38 (9212; CST), anti-phospho-p38 (4511; CST), anti-JNK (9252; CST), anti-phospho-JNK (9255; CST), anti-Gapdh (HC301; Transgen Biotech.) and anti-laminb (66095; Proteintech). The blot was detected with horseradish-peroxidase-labeled anti-rabbit (7074S; CST) or anti-mouse secondary antibody (7076S; CST) and ECL Plus solution (WBKLS0500; Millipore).
3
Fractionation and Analysis of Nuclear and Cytoplasmic Proteins
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To obtain nuclear and cytoplasmic fractions, NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific, Waltham, MA, USA) were used. Protein concentrations in the nuclear and cytoplasmic fractions were measured using a QuantiPro BCA Assay Kit (Sigma-Aldrich, St. Louis, MO, USA) and Epoch BioTek spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA). RelB and p52 NF-κB were detected in both fractions by Western blot analysis using primary anti-RelB (Abcam, Cambridge, MA, USA) and anti-NF-κB2 p100/p52 antibodies (Cell Signaling Technology, Danvers, MA, USA) and secondary goat anti-rabbit-IgG-HRP pAbs (Cell Signaling Technology, Danvers, MA, USA). Anti-GAPDH rabbit mAbs (Cell Signaling Technology, Danvers, MA, USA) were used as a cytoplasmic loading control, and anti-poly(ADP ribose) polymerase (PARP) (46D11) rabbit mAbs (Cell Signaling Technology, Danvers, MA, USA) were used as a nuclear loading control.
4
Western Blot Analysis of Cellular Proteins
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To prepare protein samples, protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Roche) were added to RIPA lysis buffer (Thermo Fisher Scientific) immediately before use. Cells were lysed in cold RIPA buffer, and the lysate concentration was measured by Bradford assay. The lysate was then diluted with loading buffer and heated at 98°C for 5 min. Proteins were resolved on NuPAGE 4‐12% Bis‐Tris gels (Thermo Fisher Scientific) based on the manufacturer’s instructions and transferred onto 0.2‐μm PVDF membranes. Blots were blocked for 1 h at room temperature in OneBlock blocking buffer (Genesee Scientific). Primary antibodies were incubated over night at 4°C. The following primary antibodies were used as follows: anti‐GAPDH (Santa Cruz sc‐365062, 1:1,000), anti‐Vinculin (Sigma‐Aldrich V9131, 1:5,000), anti‐p53 (Santa Cruz sc‐126, 1:200), anti‐p21 (Cell Signaling Technology #2947, 1:1,000), anti‐p65/RelA (Cell Signaling Technology #8242, 1:1,000), anti‐RelB (Abcam #180127, 1:1,000), anti‐Stat1 (Cell Signaling Technology #9175, 1:500), anti‐IRF3 (Cell Signaling Technology, #11904, 1:1,000), anti‐Phospho‐IRF3 (Cell Signaling Technology, #4947, 1:1,000), and anti‐ORF1p (Cell Signaling Technology #88701, 1:1,000).
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