antibodies cbp mab2676 r d systems p300 af3789 r d systems and or pcreb 9198s cell signaling technology  (R&D Systems)

Journal: Advanced Science

Article Title: The Long Non‐Coding RNA Obesity‐Related (Obr) Contributes To Lipid Metabolism Through Epigenetic Regulation

doi: 10.1002/advs.202401939

Figure Lengend Snippet: Obr associates with the Creb histone acetyltransferase complex. A) C9 cells stably overexpress, or the expression of Obr downregulated using ASOs showing the expression of Creb and histone acetyltransferases Cbp and p300. No change in the expression was observed by western blot analysis. Gapdh expression was used as the loading control. B) RNA immunoprecipitation (RIP) analysis shows that a significant amount of Obr is immunoprecipitated using an anti‐Creb antibody compared to IgG control, suggesting that Obr associates with Creb. C) Fold enrichment of Obr to that of Gapdh by RIP establishes the specificity of RIP. D) RIP analysis of C9 cells shows that Creb and histone acetyltransferases Cbp and p300 were co‐immunoprecipitated using an anti‐Creb antibody. IgG was used as control, and 10% was used as Input. E) Representative blot of ChRIP analysis of C9 cells with vector control or overexpression of Obr shows that Creb and histone acetyltransferases Cbp and p300 were co‐immunoprecipitated using an Obr antisense probe. Western analysis with pCreb, Cbp, and p300 antibodies shows all were co‐immunoprecipitated, and their level increased with over‐expression. 10% was used as input (lanes 1 and 3). The colocalization of Obr with the pCreb and histone acetyltransferases Cbp and p300 was analyzed using in situ hybridization of Obr followed by pCreb and PI as a nuclear marker F), Obr in situ, followed by pCreb and p300 immuno staining G) and H) Obr in situ followed by pCreb and Cbp immunostaining. Obr was colocalized with pCreb, p300, and Cbp in the nucleus. Scale bar = 50 µm.

Article Snippet: For RNA in situ hybridization followed by immunohistochemistry after the hybridization, the cells were incubated with primary antibodies (CBP, MAB2676, R&D systems; p300, AF3789, R&D systems; and or pCREB, 9198s, Cell Signaling Technology) followed by secondary antibodies conjugated with fluorophores (RRX Donkey Anti‐Mouse IgG, 715‐295‐151; Cy5 Donkey Anti‐Goat IgG, 705‐175‐147; Cy5 Donkey Anti‐Rabbit IgG, 711‐175‐152; RRX Donkey Anti‐Goat IgG Jackson Immuno‐Research).

Techniques: Stable Transfection, Expressing, Western Blot, Control, RNA Immunoprecipitation, Immunoprecipitation, Plasmid Preparation, Over Expression, In Situ Hybridization, Marker, In Situ, Immunostaining

Journal: Advanced Science

Article Title: The Long Non‐Coding RNA Obesity‐Related (Obr) Contributes To Lipid Metabolism Through Epigenetic Regulation

doi: 10.1002/advs.202401939

Figure Lengend Snippet: Obr is a member of the Creb histone acetyltransferase complex in vivo and is altered in diet‐induced obesity. Liver tissues from normal and HFD‐fed Wistar rats, as described in the Experimental Section, were used to assess the expression of the regulatory genes of lipid metabolism, Crebp, C/ebpβ, Pparγ, C/ebpα, Grpr, pCreb, and the acetyltransferases Cbp and P300 by western blotting. Gapdh was used as a loading control. A) The expression of these genes was increased in the livers of HFD‐fed Wistar rats compared to the chow‐fed control rats. B) ChRIP analysis using livers from the control or HFD‐fed Wistar rats shows that Creb and histone acetyltransferases Cbp and p300 were co‐immunoprecipitated using an anti‐Obr probe. Western blot analysis with pCreb, Cbp, and p300 showed all were precipitated, and their level increased in the levers of HFD‐fed animals. ChRIP analysis using a Gapdh probe was used as a negative control. 10% was used as input (lanes 1,2, 5,6, and 9,10). C–F) ChRIP analysis using the liver tissue from rats fed either with normal chow or with HFD showed increased binding to the promoters of C/ebpα, C/ebpβ, Grpr, and Pparγ. The region verified is shown above. The results show increased binding to the promoters of all these genes. Gapdh primers were used as a negative control for the ChRIP analysis.

Article Snippet: For RNA in situ hybridization followed by immunohistochemistry after the hybridization, the cells were incubated with primary antibodies (CBP, MAB2676, R&D systems; p300, AF3789, R&D systems; and or pCREB, 9198s, Cell Signaling Technology) followed by secondary antibodies conjugated with fluorophores (RRX Donkey Anti‐Mouse IgG, 715‐295‐151; Cy5 Donkey Anti‐Goat IgG, 705‐175‐147; Cy5 Donkey Anti‐Rabbit IgG, 711‐175‐152; RRX Donkey Anti‐Goat IgG Jackson Immuno‐Research).

Techniques: In Vivo, Expressing, Western Blot, Control, Immunoprecipitation, Negative Control, Binding Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inflammatory cytokines IL-32 and IL-17 have common signaling intermediates despite differential dependence on TNF-receptor 1.

doi: 10.4049/jimmunol.1002306

Figure Lengend Snippet: FIGURE 2. Phosphorylation of p300 and DAPK-1 in the presence of IL- 32 and IL-17: Human macrophage-like THP-1 cells were stimulated with either IL-32 or IL-17 (20 ng/ml each) for 15 min, followed by probing of the cell lysates in immunoblots with specific Abs to human phospho-p300 (Ser1834) or phospho–DAPK-1 (Ser308). Abs to the nonphosphorylated forms of p300 and DAPK-1 were used as paired loading control. A, An immunoblot representative of three independent experiments. B, Densitometric analysis; ratio of band density of cytokine-treated samples over unstimulated cells (y- axis), calculated after normalization to band density of nonphosphorylated forms of the respective proteins for each sample. Results represent an av- erage of three independent experiments 6 SE. *p , 0.05.

Article Snippet: Anti-human mAb directed against TNF-R1 (MAB625) and p300 Ab was obtained from R&D Systems, (Minneapolis, MN).

Techniques: Phospho-proteomics, Western Blot, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inflammatory cytokines IL-32 and IL-17 have common signaling intermediates despite differential dependence on TNF-receptor 1.

doi: 10.4049/jimmunol.1002306

Figure Lengend Snippet: FIGURE 6. Knockdown of p300 and DAPK-1 in macrophages. Human macrophage-like THP-1 cells were treated with Accell SMARTpool siRNA for either human p300, human DAPK-1, or NSC in Accell delivery media for 96 h. Knockdown efficiency was evaluated by qRT-PCR for mRNA expression (A) and immunoblots (B) by probing the nuclear extracts with specific Ab to human p300, and total cell extracts with Ab to human DAPK-1. Abs specific to human HDAC and GAPDH were used to estimate loading controls, respectively. The knockdown cells were stimulated with either IL-32 (20 ng/ml), IL-17 (20 ng/ml), IL-1b (10 ng/ml), TNF-a (10 ng/ml), or LPS (10 ng/ml) for 24 h. TC supernatants were monitored for the production of chemokine IL-8 (C) and cytokine TNF-a (D) by ELISA. Results are representative of at least three independent experiments 6 SE. *p , 0.05, **p , 0.01.

Article Snippet: Anti-human mAb directed against TNF-R1 (MAB625) and p300 Ab was obtained from R&D Systems, (Minneapolis, MN).

Techniques: Knockdown, Quantitative RT-PCR, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay