Chromatin immunoprecipitation (ChIP) analyses were performed with 1% formaldehyde-fixed U2OS or HCT116 cells using the Millipore ChIP assay kit (Cat. No. 17-295). Antibodies included normal rabbit IgG (sc-2027), anti-CUL4A (Abcam, ab92554), anti-CDT2 (Abcam, ab72264), anti-SKP2 (Abcam, ab68455), anti-H3K4me3 (Cell Signaling, 9751P) and anti-H3K9me3 (Cell Signaling, 13969P). For ChIP-Seq experiments, immunoprecipitated chromatin was sequenced and peaks were called against IgG controls by SICER using the Genomatix suite (https://www.genomatix.de/ ) and using a window size of 200 bp, gap size of 600 bp and false discovery rates (FDRs) of 0.01 for the histone marks and 0.00001 for CUL4A (p-value = 0.05). Pull-downed chromatin by CDT2 and SKP2 was sequenced and peaks were called against IgG controls by MACS2 broad peaks program. Overall, 56,715, 28,516, 12,221, 25,181, and 57,632 regions were enriched for CUL4A, CDT2, SKP2, H3K4me3, or H3K9me3, respectively.
Ab92554
Manufactured by Abcam
Sourced in United States, China
Ab92554 is a lab equipment product from Abcam. It is used for a core function in laboratory settings. No additional details can be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Duo92101, by Merck Group (2 mentions)
Ab78322, by Abcam (2 mentions)
Chip assay kit, by Merck Group (1 mentions)
Ab72264, by Abcam (1 mentions)
Anti h3k9me3, by Cell Signaling Technology (1 mentions)
H3k4me3, by Cell Signaling Technology (1 mentions)
Ab167418, by Abcam (1 mentions)
β actin ac 15, by Merck Group (1 mentions)
Ab52934, by Abcam (1 mentions)
Ab170904, by Abcam (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Ecl western blotting system, by Promega (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Prolong anti fade gold with dapi, by Thermo Fisher Scientific (1 mentions)
Mn1020b, by Thermo Fisher Scientific (1 mentions)
Ab14917, by Abcam (1 mentions)
A5971, by Merck Group (1 mentions)
Normal rabbit igg, by Cell Signaling Technology (1 mentions)
Anti h3k4me3, by Merck Group (1 mentions)
7 protocols using ab92554
1
ChIP-Seq Analysis of Chromatin Regulators
2
Protein extraction and Western blot analysis
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Protein extraction and Western blot analysis were performed as previously described with minor modifications [33 (link)]. Briefly, total cellular proteins were extracted from the harvested cells using a lysis buffer [62.5 mM Tris-HCl pH 6.8, 100 mM dithiothreitol (DTT), 2% SDS and 10% glycerol]. The protein concentrations were determined using the Bicinchonininc acid method with the Bio-Rad protein assay following the manufacturer's instructions (Bio-Rad, Hercules, CA, USA). Cellular proteins were separated on sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene fluoride membranes. Blots were incubated in blocking buffer (5% non-fat dry milk in Tris-buffered saline with 0.5% Tween, TBS-T) at room temperature for 2 h. After washing with TBS-T, the nitrocellulose was incubated with a specific antibody against MDR1 (ab170904, Abcam, CA, USA), β-actin (AC-15, Sigma Chemical Co., St. Louis, MO, USA), MRP4 (#12705, Cell Signaling, CA, USA), XPD (ab167418, Abcam, CA, USA), TOPIIA (ab52934, Abcam, CA, USA), or CUL4A (ab92554, Abcam, CA, USA) overnight at 4°C. Following the incubation with a horseradish peroxidase-conjugated secondary antibody, the signal was detected using an ECL Western Blotting System (Promega, Madison, WI, USA) and visualized and quantitated using the Bio-Rad ChemiDoc MP system.
3
Immunohistochemical Analysis of Tau Pathology
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To examine the tau pathology in brains, fixed hemi-brains were embedded in paraffin blocks and sectioned coronally at 4 μm thickness for histochemical analysis. De-paraffinized brain sections were stained with H&E or immunostained using AT8 antibody (MN1020B; Thermo Fisher Scientific), PHF-1 antibody, MC1 antibody (kind gifts from Dr. Peter Davies, Albert Einstein College of Medicine, Bronx, NY), LRP1 antibody (ab92554; Abcam), NeuN antibody (MAB377; Sigma-Aldrich), or AQP4 antibody (A5971; Sigma-Aldrich). For antigen retrieval, de-paraffinzed sections were treated with microwave (550 W, 10 min) in citrate buffer (pH 6.0) prior to immunostaining. To detect proteinase K resistant tau inclusion, sections were treated with 100 µg/ml proteinase K for 6 min at 37°C prior to AT8 and PHF-1 staining (Iba et al., 2013 (link)).
To detect tau accumulation, isolated dcLNs were embedded in 4% NuSieve GTG agarose after carefully removing fatty tissue and sliced at 100 μm thickness with a LinearSlicer PRO7 (Dosaka EM). The sections were blocked with 10% calf serum for 30 min and incubated with anti–LYVE-1 antibody (ab14917; Abcam) and washed and incubated with goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 (A-21245; Thermo Fisher Scientific). The sections were then mounted with PROLONG Anti-Fade Gold with DAPI (Thermo Fisher Scientific).
To detect tau accumulation, isolated dcLNs were embedded in 4% NuSieve GTG agarose after carefully removing fatty tissue and sliced at 100 μm thickness with a LinearSlicer PRO7 (Dosaka EM). The sections were blocked with 10% calf serum for 30 min and incubated with anti–LYVE-1 antibody (ab14917; Abcam) and washed and incubated with goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 (A-21245; Thermo Fisher Scientific). The sections were then mounted with PROLONG Anti-Fade Gold with DAPI (Thermo Fisher Scientific).
4
Chromatin Immunoprecipitation Analysis of K562 Cells
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Chromatin immunoprecipitation (ChIP) analyses were performed on 1% formaldehyde-fixed proliferating or differentiating K562 cells using a Pierce™ magnetic ChIP assay kit (Thermofisher Scientific, 26157). Antibodies used in this study included normal rabbit IgG (Cell Signaling, 2729, 1:500), normal mouse IgG (Cell Signaling, 5415, 1:500), anti-RepID (Proteintech, 20933–1-AP, 1:100), anti-CUL4A (Abcam, ab92554, 1:100), anti-JARID1A (Abcam, ab78322, 1:100), anti-H3K4me3 (Millipore, 05–1339, 1:100) and anti-H3K9me3 (Millipore, 05–1250, 1:100). For re-ChIP assays, the eluted materials from RepID precipitants were diluted with dilution buffer and subjected to the second ChIP step using antibodies against IgG, JARID1A and CUL4A. Immunoprecipitated DNA and control input DNA was analyzed by quantitative real-time PCR using the human DAB2 promoter-specific primers; forward:5’-CTC GCG GAG CTC AGG GGA G-3,’ reverse:5’-GGT AAC TCC CCC TCA ACG TG-3.’
5
Duolink in situ Protein-Protein Interaction Assay
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The Duolink in situ Red starter mouse/rabbit PLA fluorescence assay (Sigma, DUO92101) was performed according to the manufacturer’s instructions. Briefly, proliferating or PMA-induced differentiating K562 cells were attached to slides using cytospin. Cells were washed with 1 × PBS and fixed for 15 min at 4 °C in 4% paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100 in PBS for 5 min at 4 °C. The coverslips were blocked with Duolink blocking solution and incubated with CUL4A (Abcam, ab92554, 1:200) and JARID1A (Abcam, ab78322, 1:200) antibodies in Duolink antibody diluent overnight, followed by incubation with PLUS and MINUS PLA probes, ligation, and amplification. The coverslips were then washed and mounted using mounting medium containing DAPI. Images were captured using a wide-field microscope, processed, and analyzed using Zeiss Zen 3.7 and ImageJ software.
6
Proximity Ligation Assay for Protein-Protein Interactions
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The Duolink in situ Red starter mouse/rabbit PLA fluorescence assay (Sigma, DUO92101) was performed according to the manufacturer’s instructions. Briefly, proliferating or PMA-induced differentiating K562 cells were attached to slides using cytospin. Cells were washed with 1×PBS and fixed for 15 min at 4°C in 4% paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100 in PBS for 5 min at 4°C. The coverslips were blocked with Duolink blocking solution and incubated with CUL4A (Abcam, ab92554, 1:200) and JARID1A (Abcam, ab78322, 1:200) antibodies in Duolink antibody diluent overnight, followed by incubation with PLUS and MINUS PLA probes, ligation, and amplification. The coverslips were then washed and mounted using mounting medium containing DAPI. Images were captured using a wide-field microscope, processed, and analyzed using Zeiss Zen 3.7 and ImageJ software.
7
Western Blot Analysis of Cullin Proteins
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Western blotting analysis was performed as described previously [16 (link)]. Briefly, total proteins were extracted from biopsies and cultured cells using ice-cold radioimmunoprecipitation assay (RIPA) buffer (#89900, Thermo Fisher) containing 1× protease inhibitor (#87785, Thermo Fisher). Equal amounts of total proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto polyvinylidene difluoride membranes (#IPSN07852, Sigma-Aldrich). The membranes were blocked with 5% fat-free milk, followed by probing with the following primary antibodies: anti-CUL1 (#ab75817, Abcam, Shanghai, China), anti-CUL2 (#ab166917, Abcam), anti-CUL3 (#ab108407, Abcam), anti-CUL4A (#ab92554, Abcam), anti-CUL4B (#ab67035, Abcam), anti-CUL5 (#ab184177, Abcam), anti-CUL7 (#C1743, Sigma-Aldrich), anti-CUL9 (#HPA052-004, Sigma-Aldrich), anti-SKP1 (#ab76502, Abcam), anti-RBX1 (#ab133565, Abcam), anti-FBXL1 (SAB1100391, Sigma-Aldrich), anti–hypoxia-inducible factor 1α (HIF1α) (#ab2185, Abcam), anti-MEN1 (#ABC514, Sigma-Aldrich), anti-Myc (#ab32, Abcam), anti-HA (#H3663, Sigma-Aldrich), anti-Flag (#F3165, Sigma-Aldrich), and anti-GAPDH (#ab8245, Abcam). After rinsing five times with PBST buffer, the membranes were probed with secondary antibodies. Finally, protein bands were visualized using the Pierce ECL kit (#32106, Thermo Fisher Scientific).
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