Proteins were extracted using radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with a protease and phosphatase inhibitor cocktail (Merck Millipore, Burlington, MA, USA) for 30 min on ice, quantified using the Bradford protein assay (Bio-Rad), and separated on 10-12% polyacrylamide-SDS gels. Membranes were incubated overnight at 4°C with the following primary antibodies: XBP1 spliced form (NBP2-20917, 1:1000; Novus Biologicals, Centennial, CO, USA), ATF4 (11815S, 1:1000; Cell Signaling Technology, Danvers, MA, USA), ATF6 (MAB71527-SP, 1:2000; R&D Systems, Minneapolis, MI, USA), BRD4 (A301-985A; Bethyl Laboratories, Montgomery, TX, USA), KIM-1 (AF1817, 1:1000; R&D Systems), NGAL (Sc-50350, 1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and β-actin (4967S, 1:2000; Cell Signaling Technology); followed by incubation with HRP-conjugated IgG secondary antibody for 1 h. Immunostained bands were visualized using a chemiluminescence kit (Luminata Forte Western HRP Substrate; Merck Millipore), detected on an ImageLab system (Bio-Rad, Hercules, CA, USA), and quantified with ImageJ version 1.53c software (NIH Image).
Brd4 a301 985a
Manufactured by Fortis Life Sciences
Sourced in United States
Brd4 A301-985A is a laboratory equipment product. It is a research-use only item designed for the detection and study of the bromodomain-containing protein 4 (Brd4). The product's core function is to facilitate the analysis of Brd4 expression and interactions in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti flag m2 affinity gel a2220, by Merck Group (2 mentions)
H4k16ac 39167, by Active Motif (2 mentions)
H3k4me2 07 030, by Merck Group (2 mentions)
H3k36me3 07 549, by Merck Group (2 mentions)
Creb 17 600, by Merck Group (2 mentions)
His probe sc 803, by Santa Cruz Biotechnology (2 mentions)
Histone h3 9715 and h3k27me2 9755, by Cell Signaling Technology (2 mentions)
Rna pol 2 sc 899, by Santa Cruz Biotechnology (2 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Luminata forte western hrp substrate, by Merck Group (1 mentions)
Image lab system, by Bio-Rad (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Protease and phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Kim 1 af1817, by R&D Systems (1 mentions)
H3k9me2, by Merck Group (1 mentions)
Actin a5441, by Merck Group (1 mentions)
Gst sc 138, by Santa Cruz Biotechnology (1 mentions)
Acetyl histone h3, by Merck Group (1 mentions)
C myc, by Cell Signaling Technology (1 mentions)
Jmjd1a 12835 1 ap, by Proteintech (1 mentions)
6 protocols using brd4 a301 985a
1
Protein Expression Analysis Protocol
2
ChIP Experiments for Transcription Factors
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ChIP experiments were performed according to standard protocols as described (Siersbæk et al. 2012 (link)). Antibodies used were RELA (C-20, sc-372, Santa Cruz), MED1 (M-255, sc-8998, Santa Cruz), BRD4 (A301-985A, Bethyl). Sequences of primers used for qPCR are included in Supplemental Table S2B.
3
Antibody Sources for Protein Analysis
4
Chromatin Immunoprecipitation and Modification Analysis
5
Chromatin Immunoprecipitation and Modification Analysis
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LSD1 ab17721, Histone H4 ab31830, H4K20me1 ab9051, H4K20me2 ab9052, H3K79me2 ab3594, L3MBTL1 ab51880, H3K9me2 ab1220, H3K4me1 ab8895 and GST ab9085 (Abacm); ANTI-FLAG M2 Affinity Gel (A2220) (Sigma); H4K16Ac 39167 (Active Motif); H3K4me2 07-030, H3K36me3 07-549, CoREST 07-455 and CREB 17-600 (Millipore); Brd4 A301-985A (Bethyl Laboratories); H4 sc-10810, H3 sc-8654 and MEF2 sc-313, RNA Pol II sc-899 and His-probe sc-803 (Santa Cruz Biotechnology); histone H3 #9715 and H3K27me2 #9755 (Cell Signaling Technology)
6
Chromatin Immunoprecipitation in Macrophages
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BMDMs were plated in 10 cm dishes 6 Â 10 6 cells/plate. After 1 day, cells were treated with compound for 1 hour; LPS (1 mg/ml final concentration) and RVX-297 (10 mM) or DMSO (0.1%) were added, and incubations were continued for 3 hours. Chromatin immunoprecipitation (ChIP) was performed essentially as previously published (McCarthy et al., 2003; Nicodeme et al., 2010) . Briefly, cells were crosslinked with 1% formaldehyde for 10 minutes before quenching with glycine. Chromatin was sheared by sonication, and insoluble debris was removed by centrifugation. Immunoprecipitation was performed with 500 mg of protein and 2 mg of BRD antibodies (BRD2 a302-583a, BRD3 a302-368a, and BRD4 a301-985a; Bethyl Laboratories, Montgomery, TX), RNA polymerase II (polII; ab5095; Abcam), tetra-acetylated Histone H4 (06-866; Millipore, Billerica, MA), or IgG (I5006; Sigma-Aldrich). A corresponding input sample was treated equally. After de-crosslinking, DNA was isolated by phenol chloroform extraction, and real-time PCR was performed using Power SYBR Green reagent for detection (Applied Biosystems). PCR primer sequences were published by Nicodeme et al. (2010) , and the fold difference was calculated as 2^[C T (input) 2 C T (ChIP)].
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