Journal: Cell Death & Disease

Article Title: FK506 binding protein 51 positively regulates melanoma stemness and metastatic potential

doi: 10.1038/cddis.2013.109

Figure Lengend Snippet: FKBP51 interacts with the general transcriptional co-activator p300. ( a , left) FKBP51 co-immunoprecipitates with p300. ( a , right), p300 co-immunoprecipitates with FKBP51. Total-cell lysates were prepared by SAN melanoma cells transfected with FKBP51/Flag. Cell lysates were immunoprecipitated with anti-Kat3B/p300 (IP p300) or anti-Flag (IP FKBP51). Immunoprecipitated and total lysates were then subjected to western blot with anti-FKBP51 or anti-p300. ( b ) ChIP performed with SAN melanoma cells, silenced (FKBP51 siRNA) or not (NS RNA) for FKBP51. An enrichment of DNA (region at −3450 from TSS of ABCG2 gene) can be observed in p300-immunoprecipitated chromatin (NS RNA) compared with IgG sample. Such an enrichment appeared to be reduced when FKBP51 was silenced (FKBP51 siRNA)

Article Snippet: The p300- immunoprecipitated chromatin was obtained using KAT3B/p300 Antibody (RW109) (Novus Biologicals).

Techniques: Transfection, Immunoprecipitation, Western Blot

Journal:

Article Title: Repression of c-Myc and inhibition of G 1 exit in cells conditionally overexpressing p300 that is not dependent on its histone acetyltransferase activity

doi: 10.1073/pnas.1633700100

Figure Lengend Snippet: Inhibition of the S-phase entry in p300-overexpressing cells. (A) Schematic representation of the time course of serum starvation, vector infection, and harvesting of cells for FACS analysis. (B) Induction of p300 in serum-starved cells. Serum-starved cells were infected 16 h before serum stimulation with Ad vectors, labeled, and harvested at the indicated times, and p300 in the lysates was analyzed by immunoprecipitation. (C) Kinetics of S-phase entry of serum-stimulated cells. Cells were seeded, starved, and infected with Ad vectors as in A. The distribution of cells in G1, S, and G2/M phases after serum stimulation was determined by FACS analysis as described (18). The assays were done in triplicate, and the average number of cells in S phase with SD are shown.

Article Snippet: Twelve hours after infection, they were labeled with 200 uCi (1 Ci = 37 GBq) of [ 35 S]methionine per ml (3,000 Ci/mmol; Amersham Pharmacia) for 10 h, lysed in radioimmunoprecipitation assay buffer ( 28 ), and equal quantities of protein were immunoprecipitated by using an anti-p300 mAb (sc-584; Santa Cruz Biotechnology), and analyzed on SDS/8% PAGE as described ( 13 ).

Techniques: Inhibition, Plasmid Preparation, Infection, Labeling, Immunoprecipitation

Journal:

Article Title: Repression of c-Myc and inhibition of G 1 exit in cells conditionally overexpressing p300 that is not dependent on its histone acetyltransferase activity

doi: 10.1073/pnas.1633700100

Figure Lengend Snippet: Induction of p300 in rat cell clones and their growth properties. (A) Immunoprecipitation analysis of p300 (see Materials and Methods for details). Note that p300 levels for clones R12G12, 23, 48, 5, and 35 in lanes 1–10 and clones 37 and 48 cells in lanes 11–14 were assayed in separate experiments. For comparison of p300 between lanes, the levels of p300 in lanes 11–14 were normalized to clone 48 values in lanes 5–6. (B) Growth properties of rat cell clones.

Article Snippet: Twelve hours after infection, they were labeled with 200 uCi (1 Ci = 37 GBq) of [ 35 S]methionine per ml (3,000 Ci/mmol; Amersham Pharmacia) for 10 h, lysed in radioimmunoprecipitation assay buffer ( 28 ), and equal quantities of protein were immunoprecipitated by using an anti-p300 mAb (sc-584; Santa Cruz Biotechnology), and analyzed on SDS/8% PAGE as described ( 13 ).

Techniques: Clone Assay, Immunoprecipitation

Journal:

Article Title: Repression of c-Myc and inhibition of G 1 exit in cells conditionally overexpressing p300 that is not dependent on its histone acetyltransferase activity

doi: 10.1073/pnas.1633700100

Figure Lengend Snippet: Abrogation of p300-mediated S-phase inhibition by Myc. (A) Schematic representation of the time course of vector infection and harvesting of cells for FACS analysis. (B) Serum-starved cells were coinfected with Ad vectors as shown (all at 25 pfu per cell). Sixteen hours later, they were stimulated with serum and were harvested at indicated time points. Distribution of cells in G1, S, and G2/M phase were determined by FACS analysis as described (18). Adβ-gal was used where appropriate to keep the multiplicity of infection constant. Average values obtained in three independent experiments with SD are shown. For statistical validation, the mean cell numbers in lane 7 (cells infected with Adtet plus Adβ-gal) were compared with those of lane 8 (cells infected with Adtet plus Adc-Myc) by independent sample Student's two-tailed t test. P values for lane 7 vs. lane 8 for clone 48, 0.007; for clone 37, 0.011. (C) Determination of cyclin E/Cdk2 and cyclin A/Cdk2 activities in clone 37 cells at 15 h after infection with Ad vectors as shown in B.

Article Snippet: Twelve hours after infection, they were labeled with 200 uCi (1 Ci = 37 GBq) of [ 35 S]methionine per ml (3,000 Ci/mmol; Amersham Pharmacia) for 10 h, lysed in radioimmunoprecipitation assay buffer ( 28 ), and equal quantities of protein were immunoprecipitated by using an anti-p300 mAb (sc-584; Santa Cruz Biotechnology), and analyzed on SDS/8% PAGE as described ( 13 ).

Techniques: Inhibition, Plasmid Preparation, Infection, Two Tailed Test

Journal:

Article Title: Repression of c-Myc and inhibition of G 1 exit in cells conditionally overexpressing p300 that is not dependent on its histone acetyltransferase activity

doi: 10.1073/pnas.1633700100

Figure Lengend Snippet: HAT activity of p300 is not required for the inhibition of Myc- and S-phase induction. (A) Immunoprecipitation of p300 in cell clones AT21, AT41, and 37. (B) Comparison of growth properties of cell clones AT21 and AT41 with R12G12 and 37. (C) Induction of S phase in clones AT21 and AT41 with and without induction of p300. Details of this experiment were as in Fig. 2C. Assays for AT21 and AT41 were done in triplicate, for which the average values and SD are shown. (D) Determination of Myc activity in serum-stimulated clones 37, AT21, and AT41. Details are as in Fig. 3B. Values shown are luciferase activity expressed as percent of Adβ-gal controls. Averages values with SD obtained from three independent experiments are shown. (E) HAT activity of p300 in clone 37 and AT21 with induced and uninduced p300 levels. See Materials and Methods for details. Average values obtained from two experiments are shown. The radioactivity obtained from IgG incubations was subtracted from each matching sample. The values obtained for Adβ-gal infected cells were taken as 1 in calculating the fold induction.

Article Snippet: Twelve hours after infection, they were labeled with 200 uCi (1 Ci = 37 GBq) of [ 35 S]methionine per ml (3,000 Ci/mmol; Amersham Pharmacia) for 10 h, lysed in radioimmunoprecipitation assay buffer ( 28 ), and equal quantities of protein were immunoprecipitated by using an anti-p300 mAb (sc-584; Santa Cruz Biotechnology), and analyzed on SDS/8% PAGE as described ( 13 ).

Techniques: Activity Assay, Inhibition, Immunoprecipitation, Clone Assay, Luciferase, Radioactivity, Infection

Journal: Cell Death and Differentiation

Article Title: Mdm2 controls CREB-dependent transactivation and initiation of adipocyte differentiation

doi: 10.1038/cdd.2012.15

Figure Lengend Snippet: Mdm2 is required for the recruitment of Crtc2 and p300/CBP to CREs in the c/ebpδ promoter. (a) P53−/− and p53−/−;mdm2−/− MEFs were treated with vehicle or staurosporine for 4 h. C/EBPδ mRNA levels were measured using real-time PCR. (b and c) P53−/− MEFs were retrovirally transduced with vector expressing GFP or GFP-DN-Crtc. (b) Transduced MEFs were treated with vehicle or IBMX for 1 h. mRNA levels were determined using real-time PCR. (c) Transduced MEFs were induced to undergo adipogenesis according to the MDI standard protocol in the presence of rosiglitazone. Adipocyte differentiation was scored by adipocyte marker gene expression. GLUT4, glucose transporter 4. (d) In vitro translated Crtc2 was pulled down using Mdm2 or its halves fused to GST. (e) At 2 days postconfluence, p53−/− and p53−/−;mdm2−/− MEFs were left untreated or stimulated with IBMX for 30 min. Binding of P-CREB, Crtc2, p300 and CBP to the c/ebpδ promoter was assessed by chromatin immunoprecipitation. Non-specific IgG was included as control. β-Globin was used to assess background levels

Article Snippet: Antibodies used were P-CREB-1 (Ser133) (sc-7978), CBP (sc-369), p300 (sc-584) and Crtc2 (sc-46272) (all from Santa Cruz Technology, Santa Cruz, CA, USA).

Techniques: Real-time Polymerase Chain Reaction, Transduction, Plasmid Preparation, Expressing, Marker, Gene Expression, In Vitro, Binding Assay, Chromatin Immunoprecipitation, Control

Journal: Cell proliferation

Article Title: Elevated COMMD1 Contributes to Cardiomyocyte Copper Efflux in Chronic Myocardial Ischemia: Insights From Rhesus Monkey.

doi: 10.1111/cpr.70016

Figure Lengend Snippet: FIGURE 3 | Increased XIAP associated with COMMD1 in cardiomyocytes after chronic LAD ligation in rhesus monkeys. (A) The mRNA levels of COMMD1 determined by RT-PCR. (B) Western blot and (C) quantitative analysis of the protein levels of p300 and XIAP in different portions of the heart of rhesus monkey (Sham: n = 4; MI: n = 4). (D) Immunofluorescence image and (E) co-localization analysis showing COMMD1 (green) or XIAP (green) with cardiomyocytes (α-actinin, red) in the serial sections of the heart of rhesus monkey, DAPI stain (blue) labels nuclei, scale bar = 100 μm. (F) Immunoprecipitation-Western blotting analysis of COMMD1 and XIAP in the ischemic heart tissue of rhesus monkeys and the control res- in + mouse antibody against COMMD1 or coupling resin + mouse IgG as the control groups. (G) Western blotting analysis of the protein levels of COMMD1 and XIAP in the cardiac tissues of rhesus monkeys after 2 and 24 h of LAD ligation (Sham: n = 4; 2 h MI: n = 2; 24 h MI: n = 2). (H) The ratio of XIAP versus COMMD1 protein levels in the heart of rhesus monkeys. Data were expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 versus Sham control. IA, infarct area; RA, remote area.

Article Snippet: PVDF membranes were incubated overnight at 4°C with the respective primary antibodies: monoclonal mouse anti- CCS (1:100, SC- 55560, Santa cruz, USA), monoclonal mouse anti- COMMD1 (1:100, SC- 166248, Santa cruz, USA), polyclonal rabbit anti- COMMD1 (1:1000, A5174, Abclonal, China), monoclonal mouse anti- XIAP (1:100, SC- 55551, Santa Cruz, USA), polyclonal rabbit anti- XIAP (1:1000, NB- 100- 56,183, Novus, USA), monoclonal rabbit antiATP7A (1:1000, ab308524, abcam, USA), monoclonal rabbit anti- ATP7B (1:1000, ab131208, abcam, USA), monoclonal mouse anti- GAPDH (1:1000, TA- 08, Zhongshan, China) and monoclonal rabbit anti- p300 (1:1000, 86,377, CST, USA).

Techniques: Ligation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunofluorescence, Staining, Immunoprecipitation, Control

Journal: Biomedicines

Article Title: R -α-Lipoic Acid and 4-Phenylbutyric Acid Have Distinct Hypolipidemic Mechanisms in Hepatic Cells

doi: 10.3390/biomedicines8080289

Figure Lengend Snippet: PBA enhances the acetylation of CBP/p300 and histone H3 at the CPT1A and INSIG2 promoters in Huh7-shTSC2 cells. PBA (8 mM, 6 h) induced the gene expression of CPT1A and INSIG2 ( A ), and the acetylation status of histone H3 and histone H4 ( B ). Chromatin immunoprecipitation (ChIP) assay revealed PBA-mediated enrichment of acetyl CBP/p300 (Ac-p300) and acetyl histone H3 (Ac-H3) at the CPT1A and INSIG2 promoters ( C ). Statistical significance was determined using Student’s t -test, * p < 0.05, n = 4.

Article Snippet: After pre-clearing the sheared chromatin, samples were subjected to immunoprecipitation using the following the antibodies: anti-acetyl-CBP [Lys1535]/p300 [Lys1499] (Cell Signaling Technology #4771S, Danvers, MA, USA), anti-acetyl-histone H3 (Millipore #06–599, Burlington, MA, USA), anti-histone H3 (Millipore #07–690).

Techniques: Gene Expression, Chromatin Immunoprecipitation

Journal: Advanced Science

Article Title: Chemotherapy‐Mediated Induction of PD‐L1 via SEI1 Facilitates Myeloma Immune Evasion

doi: 10.1002/advs.202411082

Figure Lengend Snippet: SEI1 bridges the interaction between CBP/p300 and PAF1 complex to regulate PD‐L1 transcription. A) HA‐SEI1 overexpressed IM‐9 cells were lysed, and the total protein lysate was immunoprecipitated with an agarose‐immobilized HA antibody and analysis with mass spectrometry. The proteins identified are indicated. B,C) Co‐immunoprecipitation of SEI1, PAF1, CDC73, or p300 in ARH‐77 or IM‐9 cells treated with melphalan or bortezomib. D) Co‐immunoprecipitation of SEI1 along with PAF1, CDC73, or p300 in HEK293T cells co‐transfected with SEI1 and either PAF1 , CDC73 , or p300 plasmid. E) Schematic of the truncations including ΔCABD, ΔSD, ΔPBID, and ΔCT fragments. F) Western blotting showing different truncations of SEI1 (full length, ΔCABD, ΔSD, ΔPBID, and ΔCT) in HEK293T cells. G–K) Pulldown of PAF1, CDC73, or p300 with different truncations of SEI1 (full length, ΔCABD, ΔSD, ΔPBID, and ΔCT) in HEK293T cells. L) Pulldown of MYC‐p300 with CDC73 or PAF1 in HEK293T cells. M) Gene tracks showing representative ATAC‐seq profiles at PD‐L1 gene loci in primary CD138 + plasma cells from two relapsed myeloma patients following treatment with bortezomib. N,O) ChIP PCR assay showing SEI1, p300, or H3K27ac enrichment on PD‐L1 promoter of ARH‐77 or IM‐9 cells treated with or without melphalan or bortezomib. P) ChIP‐seq profiles show H3K27ac signal tracks at the PD‐L1 gene loci for ARH‐77 cells treated with or without melphalan or bortezomib. Q) ChIP PCR assay showing H3K27ac enrichment on PD‐L1 promoter of ARH‐77 or IM‐9 cells treated with or without melphalan or bortezomib. R) ChIP PCR assay showing enrichment of H3K27ac on PD‐L1 promoter in sh Ctrl or sh SEI1 ARH‐77 or IM‐9 cells. S) Expression of PAF1 in nonspecific, PAF1 shRNA‐expressing myeloma cells. T,U) The relative expression of PD‐L1 in PAF1 knockdown ARH‐77 and IM‐9 cells treated with or without melphalan or bortezomib. V,W) ChIP PCR assay showing enrichment of V) SEI1 or W) H3K27ac on PD‐L1 promoter in sh Ctrl or sh PAF1 ARH‐77 and IM‐9 cells. X) Schematic of the SEI1 bridges the interaction between CBP/p300 and PAF1 complex to regulate PD‐L1 transcription. B–D,F–L,N,O,Q–W) Representative of three independent experiments. Data are averages ± SD. *** p < 0.001 and **** p < 0.0001. All p ‐values were determined using one way ANOVA.

Article Snippet: Chromatin samples were immunoprecipitated with antibodies against phosphorylated IRF7 (#12390), acetyl‐histone H3 (Lys27) (H3k27ac) (#8173), p300 (#54062), phosphorylated p65 (#3033), trimethyl‐histone H3 (Lys4) (H3K4me3) (#9751), trimethyl‐histone H3 (Lys27) (H3k27me3) (#9733), trimethyl‐histone H3 (Lys36) (H3k36me3) (#4909), phosphorylated IRF3 (#29 047) (Cell Signaling Technology), SEI1 (sc‐517080, Santa Cruz), and control IgG at 4 °C for 3 h. Immunoprecipitates and total chromatin inputs were reverse cross‐linked; DNA was isolated and analyzed using PCR with primers.

Techniques: Immunoprecipitation, Mass Spectrometry, Transfection, Plasmid Preparation, Western Blot, Clinical Proteomics, ChIP-sequencing, Expressing, shRNA, Knockdown