Bone marrow was isolated from C57 mice and differentiated for 7 days using media containing M-CSF to generate bone marrow-derived macrophages (BMDMs) as described previously (Link et al., 2018a (link)). BMDMs were maintained at basal conditions or treated with KLA for 1 h. For p65 (Santa Cruz, sc-372X) and p50 (Abcam, ab32360) ChIP-seq experiments, 8 million untreated or KLA-treated BMDMs per assay were double-crosslinked using disuccinimidyl glutarate and formaldehyde (FA). ChIP-seq was performed using 2 μg of antibody as described previously (Heinz et al., 2018 (link)). ChIP DNA was prepared for sequencing using the NEBNext Ultra II DNA library prep kit (NEB, E7645) and sequencing was performed on the HiSeq4000 (75 bp SR, Illumina). The binding sites of p65 and p50 were identified using HOMER ‘findPeaks -size 200’ (Heinz et al., 2010 (link)) and then merged to obtain co-binding sites and p65- or p50-only binding sites. The binding activity of p65 and p50 was quantified by the count of ChIP-seq reads. The raw and processed data have been deposited to the NCBI GEO under the accession number GSE144070.
Sc 372x
Manufactured by Abcam
Sc-372X is a laboratory equipment product. It is designed to perform a core function within a laboratory setting. However, a detailed description of its specific features and capabilities cannot be provided in an unbiased and factual manner without the risk of extrapolation or interpretation. Therefore, a comprehensive description of this product is not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab32360, by Abcam (2 mentions)
Hiseq 4000, by Illumina (2 mentions)
Nebnext ultra 2 dna library prep kit, by New England Biolabs (2 mentions)
Phospho stat3 tyr705, by Cell Signaling Technology (1 mentions)
H3k27ac, by Cell Signaling Technology (1 mentions)
Icbp112, by Merck Group (1 mentions)
Cytokine, by R&D Systems (1 mentions)
Bp 1 102, by Merck Group (1 mentions)
Sc 482x, by Abcam (1 mentions)
Sc 150x, by Abcam (1 mentions)
Sgc cbp30, by Merck Group (1 mentions)
Ab62739, by Abcam (1 mentions)
α actin, by Merck Group (1 mentions)
α flag, by Merck Group (1 mentions)
α tubulin, by Merck Group (1 mentions)
α acetyl lysine, by Cell Signaling Technology (1 mentions)
αh3k9me3, by Merck Group (1 mentions)
αh3k9ac, by Cell Signaling Technology (1 mentions)
α p iκb α, by Cell Signaling Technology (1 mentions)
Sc 371, by Cell Signaling Technology (1 mentions)
4 protocols using sc 372x
1
ChIP-seq analysis of NF-κB subunits
2
Cytokine-Induced Gene Regulation Assay
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Cytokines (R&D systems) were reconstituted in PBS 0.1% BSA. Treatment durations and final concentrations were optimized and set at 2 h for the lowest concentrations leading to maximal gene induction (IL-1β and IL-6 at 10 ng ml−1, TNF at 2 ng ml−1). p300/CBP inhibitors C646, SGC-CBP30 and I-CBP112 (Sigma, Cat# SML0002, SML1133 and SML1134 respectively) were reconstituted in DMSO and treated at a final concentration of 35 µM 20 min. prior to cytokine treatment. STAT3 inhibitor (BP-1-102, Sigma, Cat# 573132) was reconstituted with DMSO and treated at a final concentration of 30 µM 20 min. prior to cytokine treatment. Adenoviral vectors targeting AP-1, NF-κB and CEBPB (Vector Biolabs, Cat# 1046, 1028 and shADV-255244, respectively) were added to hepatocytes 4 h after plating (18 h prior to cytokine treatments) at a final concentration of 106 pfu per 35 mm well (for qPCR) or 1006 pfu per 15 cm plate (for ChIP)
Antibodies used: STAT3, p65 and CEBPB (Santa Cruz biotechnology, sc-482X, sc-372X, sc-150X, respectively), GAPDH and RNAP II (Abcam, Cat# ab8245 and ab5131, respectively), cJun and phospho-STAT3 Tyr705 (Cell Signaling Technologies, Cat# #9165 and #9145, respectively) and H3K27ac (Active motif #39133).
Antibodies used: STAT3, p65 and CEBPB (Santa Cruz biotechnology, sc-482X, sc-372X, sc-150X, respectively), GAPDH and RNAP II (Abcam, Cat# ab8245 and ab5131, respectively), cJun and phospho-STAT3 Tyr705 (Cell Signaling Technologies, Cat# #9165 and #9145, respectively) and H3K27ac (Active motif #39133).
3
Antibody Characterization for Western Blot and ChIP-seq
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The antibodies used were α-HA (Sigma-Aldrich H6908; WB 1 : 1,000), α-FLAG (Sigma-Aldrich F7425, WB 1 : 1,000 ChIP 5 μg), α-myc (Cell Signaling 2276 S, WB 1 : 5,000 immunofluorescence (IF) 1:150 ChIP 5 μg), α-H3K9me3 (Millipore 07–442, WB 1 : 1,000; Abcam ab8898, IF 1 : 150 ChIP 5 μg), α-H3K9ac (Cell Signaling ab1191, ChIP 5 μg), α-actin (Sigma-Aldrich A1978, WB 1 : 5,000), α-tubulin (Sigma-Aldrich T6199, WB 1 : 20,000), α-NFkB p65 (Santa Cruz Biotechnology sc-372-x, WB 1 : 1,000), α-SirT6 (AbCam ab62739, WB 1 : 1,000; ChIP 5μg), α-H3 (Cell Signaling 9715 S, WB 1 : 2,000), α-acetyl-lysine (Cell Signaling 9814 S, WB 1 : 1,000), α-suv39h1(Millipore 07–550, WB 1 : 1,000), α−SKP2 (Thermo Fisher Scientific 32–3300, WB 1 : 1,000), α-IκBα (Santa Cruz sc-371, 1 : 1,000), α-P-IκBα (Cell Signaling 9246 S, WB 1 : 1,000), and α-p100/p52 (Millipore 05-361, WB 1 : 1,000).
WBs were performed as described elsewhere. Images of original WBs included in the main figures are included in Supplementary Figure9 .
WBs were performed as described elsewhere. Images of original WBs included in the main figures are included in Supplementary Figure
4
NF-κB Subunit Binding Profiling in BMDMs
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Bone marrow was isolated from C57 mice and differentiated for 7 days using media containing M-CSF to generate bone marrow-derived macrophages (BMDMs) as described previously (Link et al., 2018a) . BMDMs were maintained at basal conditions or treated with KLA for 1 hour. For p65 (Santa Cruz, sc-372X) and p50 (Abcam, ab32360) ChIP-seq experiments, 8 million untreated or KLA-treated BMDMs per assay were double-crosslinked using disuccinimidyl glutarate (DSG) and formaldehyde (FA). ChIP-seq was performed using 2 µg of antibody as described previously (Heinz et al., 2018) . ChIP DNA was prepared for sequencing using the NEBNext Ultra II DNA library prep kit (NEB, E7645) and sequencing was performed on the HiSeq4000 (75bp SR, Illumina). The binding sites of p65 and p50 were identified using HOMER "findPeaks -size 200" (Heinz et al., 2010) and then merged to obtain co-binding sites and p65-or p50-only binding sites. The binding activity of p65 and p50 was quantified by the count of ChIP-seq reads.
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