Cells and tissues were lysed by homogenization in RIPA lysis buffer (ThermoFisher Scientific, #89900). Protein extracts were separated by SDS-PAGE and then transferred onto PVDF membrane (Millipore, #IPVH00010). Following overnight incubation with the indicated primary antibodies at 4°C, membranes were incubated with IR dye-coupled secondary antibodies (LI-COR) and then visualized by using the LI-COR Odyssey infrared imaging system (LI-COR). The primary antibodies and diluted ratio include: anti-phospho-AKT 1:1000 (Ser473; Cell Signaling Technology, #9271); anti-AKT 1:1000 (Cell Signaling Technology, #2920); anti-β-ACTIN 1:5000 (Sigma, #A1978); anti-IκBα 1:1000 (Cell signaling Technology, #4814); anti-α-Tubulin 1:1000 (Cell Signaling Technology, #3873); anti-Acetylated Lysine 1:1000 (Cell Signaling Technology, #9441); anti-NFκB p65 1:1000 (Cell Signaling Technology, #8242); anti-p300 1:1000 (Cell Signaling Technology, #86377); anti-FLAG (Sigma, #F1804); anti-Acetyl NFκB p65 (Lys310) 1:1000 (Cell Signaling Technology, #12629). NuRD complex components (CHD4, MTA1, HDAC1, RbAP46) antibodies were all from NuRD Complex Antibody Sampler Kit (Cell Signaling Technology, #8349) and diluted 1:1000 for primary antibody incubation. Detailed information of commercial manufacturers and validation of the antibodies can be found in the Reporting Summary.
Anti p300
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-p300 is a lab equipment product that targets the p300 protein. p300 is a histone acetyltransferase that plays a role in gene regulation and transcription. Anti-p300 can be used to detect and study the p300 protein in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Merck Group (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Anti phospho akt, by Cell Signaling Technology (2 mentions)
Anti nf κb p65, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Anti flag, by Merck Group (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti acetylated lysine, by Cell Signaling Technology (2 mentions)
Anti α tubulin, by Cell Signaling Technology (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Anti acetyl nfκb p65 lys310, by Cell Signaling Technology (2 mentions)
Nurd complex antibody sampler kit, by Cell Signaling Technology (2 mentions)
Ir dye coupled secondary antibodies, by LI COR (2 mentions)
Anti iκbα, by Cell Signaling Technology (2 mentions)
Anti actin, by Merck Group (2 mentions)
Anti src 1, by Cell Signaling Technology (2 mentions)
Anti glucocorticoid receptor, by Cell Signaling Technology (2 mentions)
Hek293t cell, by American Type Culture Collection (1 mentions)
Anti hsp90, by Cell Signaling Technology (1 mentions)
Control igg, by Cell Signaling Technology (1 mentions)
13 protocols using anti p300
1
Western Blot Analysis of Cell Signaling
2
Cell Culture and Authentication Protocol
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MDA-MB-468 and MDA-MB-231 were obtained from Developmental Therapeutics Program at the National Cancer Institute. HEK293T cells were obtained from ATCC. All the cells were authenticated by STR profiling using Promega 10 GenePrint assay. The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and non-essential amino acids in a humidified incubator at 37 °C with 5% CO2. The cells were used until passage 50. The following antibodies were used: anti-TPM (Sigma, cat #T2780), anti-Hsp10 (Bethyl laboratories, cat # A304-842A-M), anti-Hsp90 (cat # 4874) and anti-P300 (cat # 54,062) (Cell Signaling Technology). Secondary antibodies were from Jackson ImmunoResearch Laboratories.
3
ChIP-qPCR Analysis of Smad2/3 and p300 Binding
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HCCLM3 cells (3 × 106 cells/10-cm plate) were harvested for the ChIP assay. Briefly, cells were washed with PBS and cross-linked with 1% formaldehyde for 10 min at room temperature. The reaction was stopped by adding 1.25 M glycine for 5 min at room temperature. After washing, nuclear lysis, and sonication, the chromatin was sheared into 250–800 bp fragments. Then, the chromatin fraction was incubated with anti-Smad2/3 (#8685S, Cell Signaling Technology) and anti-p300 (#86377S, Cell Signaling Technology) antibodies or the control IgG (#3900S, Cell Signaling Technology) overnight at 4°C. Chromatin-bound beads were subjected to extensive washing, and the eluted chromatin was de-cross-linked using ChIP elute buffer with proteinase K for 2 h at 65°C. The eluted DNA was purified for subsequent q-PCR analysis. The q-PCR primers were as follows: TGF-β1 forward (–462), 5′-GTCCTGTTGCCCCCTCCT-3′; TGF-β1 reverse (–378), 5′-CCCAGAACGGAAGGAGAGTC-3′; SOX4 forward (+427), 5′-AGCTTCAGCAACCAGCATTC-3′; and SOX4 reverse (+512), 5′-CCCTCTCTCTCGCTCTCTCA-3′.
4
Immunohistochemical Analysis of Cellular Proteins
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Sections were incubated with the following primary antibodies: anti-PanKbu (mouse polyclonal antibody; 1 : 200 dilution; Hangzhou Jing Jie biological Co., Ltd; China), anti-H3K9bu (mouse polyclonal antibody; 1 : 200 dilution; Hangzhou Jing Jie biological Co., Ltd; China), anti-Fn (rabbit polyclonal antibody; 1 : 200 dilution; Abcam; UK), and anti-P300 (rabbit polyclonal antibody; 1 : 200 dilution; Cell Signaling Technology; USA) overnight at 4°C. After sections were washed with PBS, they were incubated with horseradish peroxidase (HRP) or fluorescein isothiocyanate fluorescent dye-conjugated secondary antibodies (1 : 200 dilution; Beijing Biosynthesis Biotechnology; China) for 2 h at room temperature. For visualizing the signals of immunohistochemistry, sections were treated with peroxidase substrate 3,3-diaminobenzidine and counterstained with hematoxylin. Each photograph of the stained sections was scanned using a light microscope.
5
Cell Lysis and Western Blot Analysis
6
Immunohistochemical Analysis of p300 Expression in Prostate Cancer
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For IHC staining, a tissue microarray (TMA) of 14 patients that received neoadjuvant docetaxel therapy before radical prostatectomy and 14 patients with no chemotherapy was used. The use of patient material was approved by the Ethics Committee of the Medical University of Innsbruck (study No AM 3174 including amendment 2). For detailed information about clinical data from patients, see publication of Puhr et al. (2012) (link). IHC staining was performed on a Discovery-XT staining device (Ventana) and the following specific antibody was used: anti-p300 (1:100, D8Z4E, Cell Signaling Technology). Antibody specificity was verified by Western blot and IHC staining of PC3-DR cells with p300 downregulation. For IHC, cells were embedded by coagulation in plasma clots after harvesting, transferred into a biopsy histosette, fixed in formalin, and embedded in paraffin. Importantly, cross staining of CBP was excluded by Western blot analysis.
7
Comprehensive Antibody Panel for Protein Analysis
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The following antibodies were used in the experiments: mouse anti‐glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (G8795, 1:10,000) and anti‐Flag (F3165, 1:2000) from Sigma‐Aldrich (St. Louis, MO, USA); mouse anti‐Myc (11667149001, 1:2000) and mouse anti‐hemagglutinin (HA) antibody (11583816001, 1:2000) from Roche Applied Science (Penzberg, Germany); rabbit anti‐Myc (562, 1:2000), rabbit anti‐HA antibody (561, 1:2000), and anti‐Strep‐tag II (M211‐3, 1:3000) from MBL Life Science (MBL, Nagoya, Japan); anti‐HIF2α (NB100‐122, 1:1000) from Novus Biologicals (Centennial, CO, USA); anti‐histone H3 (acetyl K27) (H3K27ac, 1:100 for ChIP) (ab4729) from Abcam (Cambridge, MA, USA); anti‐HDAC1 (GTX100513) from GeneTex (Irvine, CA, USA); rabbit anti‐junction plakoglobin (JUP) (#75550, 1:1000) and anti‐p300 (#86377, 1:100 for ChIP) from Cell Signaling Technology (Danvers, MA, USA); mouse anti‐JUP (#MA5‐15905, 1:1000), goat anti‐mouse IgG secondary antibody (31430, 1:20,000), and goat anti‐rabbit IgG‐horseradish peroxidase (HRP) secondary antibody (31460, 1:20,000) from Thermo Fisher Scientific (Waltham, MA, USA).
8
Comprehensive Protein Analysis Protocols
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Immunoprecipitations, Western blot analysis, and immunoprecipitation Westerns were carried out by standard methods described before (18 (link), 25 (link)). Densitometry was performed using Scion Image software (Scion Corp., Frederick, MD). Antibodies were obtained from commercial sources listed below. Anti-PP2Cδ, anti-ATM, anti-ATM (pS1981), anti–caspase-3, horseradish peroxidase (HRP)–anti-mouse immunoglobulin G (IgG), and HRP–anti-rabbit IgG were obtained from Santa Cruz Biotechnology; anti-p53, anti-p53 (acetyl K382), anti-p53 (acetyl K373), and anti-BRCA1 (pS1423) were provided by Abcam; and anti-BRCA1, anti-p300, anti–cleaved caspase-3, anti-actin, and anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology.
9
Antibody Sources for Protein Analysis
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Specific antibodies were purchased from the following commercial sources: Anti-AFP, anti-ALB, anti-CD44, anti-Evi1, anti-flag (mouse), anti-HA, anti-HNF4α, anti-H3, anti-H3K36me3, anti-Myc, anti-OCT4A, anti-P300, anti-PPM1A (rabbit), anti-pSmad2 (S465/467), anti-pSmad2 (S245/250/255), anti-pSmad3 (s423/425), anti-Smad2, anti-Smad3 (rabbit), anti-SnoN, anti-Sox2, anti-TAT, and normal rabbit IgG from Cell Signaling Technology (Danvers, MA); anti-PPM1A (mouse), anti-SC35, and anti-SETD2 were from Abcam (Cambridge, MA); Anti-Smad4 and normal mouse IgG were from Santa Cruz Biotechnology (Santa Cruz, CA); Anti-β-actin, and anti-flag (rabbit) from Sigma-Aldrich (St. Louis, MO); Alexa594 goat anti-mouse IgG from Life Technology (Carlsbad, CA); Dylight488 goat anti-rabbit IgG from Vector Labs (Burlingame, CA).
10
Co-IP of XBP1s and p300 in Hypoxic Cells
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RAW264.7 cells were randomly assigned into normal control, hypoxia, hypoxia +0.1% DMSO (vehicle) and hypoxia +L002 (p300 inhibitor; 5 μmol/L for 24 hours) groups. The Catch and Release® v2.0 reversible immunoprecipitation system (#17‐500, Millipore, USA) was used for co‐IP. The cells were lysed, after which 2 μg/mL anti‐XBP1s (#40435) or anti‐p300 (#86377) antibodies purchased from Cell Signalling Technology were used to precipitate the proteins. Mouse anti‐rabbit IgG (#3678, Cell Signalling Technology; 1:1000) antibody was used as a negative control. The precipitated proteins were resolved by SDS‐PAGE and transferred to PVDF membranes. The membranes were incubated with primary antibodies against Ac‐K, XBP1s, p300 and GAPDH overnight at 4℃, followed by incubation with HRP‐conjugated goat‐anti‐rabbit IgG (31460, Invitrogen, USA; 1:1000), and the signals werer developed using the ECL method.
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