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: Clinical and Translational Medicine

Article Title: FKBP51 increases the tumour-promoter potential of TGF-beta

doi: 10.1186/2001-1326-3-1

Figure Lengend Snippet: FKBP51 interacts with the general transcriptional co-activator p300 and the TGF-β transcription factor Smad2/3. Left; FKBP51 co immunoprecipitates with p300. 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, anti-p300 or anti-Smad 2/3. Smad 2/3 co immunoprecipitate with either p300 (left) and FKBP51 (right).

Article Snippet: Three μg anti-KAT3B/p300 (Novus Biologicals, Littleton, CO, USA) or anti-FKBP51 (H100, rabbit polyclonal, Santa Cruz Biotechnology), was added to total lysate, kept in rotation, at 4°C over night.

Techniques: Transfection, Immunoprecipitation, Western Blot

Journal: Clinical and Translational Medicine

Article Title: FKBP51 increases the tumour-promoter potential of TGF-beta

doi: 10.1186/2001-1326-3-1

Figure Lengend Snippet: Mechanism proposed for FKBP51 enhancement of TGF-β pro-oncogenic signal. Left, FKBP51 facilitates Smad recruitment to coactivators. Right, FKBP51 takes part to the transcriptional complex formed by P300 and Smad 2,3 Increase in FKBP51, as it occurs in melanoma, generates an auto regulatory loop of TGF-β signaling, which in turn promotes tumour progression.

Article Snippet: Three μg anti-KAT3B/p300 (Novus Biologicals, Littleton, CO, USA) or anti-FKBP51 (H100, rabbit polyclonal, Santa Cruz Biotechnology), was added to total lysate, kept in rotation, at 4°C over night.

Techniques:

Journal: American journal of physiology. Endocrinology and metabolism

Article Title: The transcriptional coregulator CITED2 suppresses expression of IRS-2 and impairs insulin signaling in endothelial cells.

doi: 10.1152/ajpendo.00435.2020

Figure Lengend Snippet: Figure 1. CITED2 selectively inhibits insulin signaling in endothelial cells in vitro and in vivo. A–E: lung endothelial cells were isolated from Cited2fl/flmice and treated with adenovirus expressing cre recombinase (Ad-Cre) or GFP only (Ad-GFP), thereby creating CITED2 knockout (CITED2 KO) and control cultures from the same cell isolation. A: CITED2 expression was measured in cell lysate by Western blotting. B: cell cultures were serum-starved over- night and treated with 10 nM insulin for 5 min. Protein expression was measured in cell lysate by Western blotting. C–E: quantitative analysis of insulin- stimulated phosphorylation of insulin receptor-b (INSRb), Akt, and ERK based on densitometry of 3–4 independent experiments and normalization to the baseline phosphorylation in control cells. F–G: Cdh5-cre Cited2fl/fland Cited2fl/flcontrol mice were injected intravenously with 5 U insulin or vehicle. F: after 5 min, the aorta was isolated and flash-frozen. Levels of Akt, phosphorylated Akt, and b-actin were measured by Western blotting of tissue lysate. G: quantitative analysis of insulin-stimulated Akt phosphorylation based on densitometry of 5 pairs of animals. P < 0.05, P < 0.01, P < 0.001, 2- way ANOVA followed by Tukey’s multiple-comparisons test. CITED2, CBP/p300 interacting transactivator-2; ERK, extracellular signal-related kinase; GFP, green fluorescent protein.

Article Snippet: Chromatin Immunoprecipitation Chromatin immunoprecipitation (ChIP) was performed as previously described (15) with an anti-p300 antibody (NB100616, Novus Biologicals).

Techniques: In Vitro, In Vivo, Isolation, Expressing, Knock-Out, Control, Cell Isolation, Western Blot, Phospho-proteomics, Injection

Journal: American journal of physiology. Endocrinology and metabolism

Article Title: The transcriptional coregulator CITED2 suppresses expression of IRS-2 and impairs insulin signaling in endothelial cells.

doi: 10.1152/ajpendo.00435.2020

Figure Lengend Snippet: Figure 4. CITED2 overexpression recapitulates endothelial insulin resistance. A–C: MS1 endothelial cells were cultured with lentivirus expressing FLAG- CITED2 for 12 h, and then, lentiviral medium was replaced with complete medium for 48h. Stably transduced MS1 cells were selected with puromycin (0.1%) for 72h. A: control and FLAG-CITED2-overexpressing cell cultures were serum-starved overnight and treated with 10 nM insulin for 5 min. Protein levels were measured in cell lysate by Western blotting. B: quantitative analysis of insulin-stimulated phosphorylation of Akt based on densitometry of 4 independent experiments. C: control and FLAG-CITED2-overexpressing cultures were serum-starved overnight and treated with 10 nM insulin for 4 h. Vegfa expression was measured by real-time PCR of 3 independent experiments, and relative Vegfa mRNA expression was determined by normaliza- tion to the housekeeping gene Rplp0. P < 0.05, P < 0.01, 2-way ANOVA followed by Tukey’s multiple-comparisons test. CITED2, CBP/p300 interact- ing transactivator-2.

Article Snippet: Chromatin Immunoprecipitation Chromatin immunoprecipitation (ChIP) was performed as previously described (15) with an anti-p300 antibody (NB100616, Novus Biologicals).

Techniques: Over Expression, Cell Culture, Expressing, Stable Transfection, Control, Western Blot, Phospho-proteomics, Real-time Polymerase Chain Reaction

Journal: American journal of physiology. Endocrinology and metabolism

Article Title: The transcriptional coregulator CITED2 suppresses expression of IRS-2 and impairs insulin signaling in endothelial cells.

doi: 10.1152/ajpendo.00435.2020

Figure Lengend Snippet: Figure 5. CITED2 limits HIF-2a-mediated transcription of Irs2. A: MS1 endothelial cells transduced with lentivirus expressing FLAG-CITED2 were cotrans- fected with Renilla luciferase and normoxia-stable pcDNA3.1 mHIF-2a MYC P405A/P530V/N851A (HIF2a-DNA3.1), Irs2 promoter reporter (Irs2-pGL3), or empty vector control (pGL3, pcDNA3.1) plasmids. After 24h, cells were lysed and luminescent signal measured with the Dual Luciferase Reporter Gene Assay kit. B: CITED2 KO and control endothelial cells were cross-linked, lysed, and sonicated to produce 200–1,000-bp chromatin fragments as ana- lyzed by agarose gel electrophoresis. Immunoprecipitations were performed with 10 μg of p300 antibody (NB100-616, Novus Biologicals) or mouse IgG. Relative enrichment of chromatin was determined by real-time PCR. C: CITED2 inhibition of the p300/CBP-HIF-2a transcriptional complex decreases Irs2 transcription and IRS-2 expression, leading to impaired insulin signaling in endothelial cells. P < 0.01, Student’s t test. P < 0.001, P < 0.0001, 1-way ANOVA followed by Tukey’s multiple-comparisons test. CBP, CREB-binding protein; CITED2, CBP/p300 interacting transactivator-2; ChIP, chromatin immunoprecipitation; HIF, hypoxia-inducible factor; HRE, hypoxia-response element; INSR, insulin receptor.

Article Snippet: Chromatin Immunoprecipitation Chromatin immunoprecipitation (ChIP) was performed as previously described (15) with an anti-p300 antibody (NB100616, Novus Biologicals).

Techniques: Transduction, Expressing, Luciferase, Plasmid Preparation, Control, Reporter Gene Assay, Sonication, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Inhibition, Binding Assay, Chromatin Immunoprecipitation