Journal: Immunology

Article Title: SHARPIN controls the development of regulatory T cells

doi: 10.1111/imm.12604

Figure Lengend Snippet: T‐cell receptor (TCR) ‐mediated nuclear factor‐κB (NF‐κB) and c‐Jun N‐terminal kinase (JNK) activation is reduced in SHARPIN‐deficient thymocytes. Thymocytes isolated from Sharpin +/+; Foxp3 gfp/gfp and Sharpin cpdm/cpdm; Foxp3 gfp/gfp mice were left untreated (co) or stimulated with anti‐CD3/anti‐CD28 (CD3/28) or PMA/ionomycin (P/I) for the indicated time‐points. (a) Total cell lysates were analysed by immuno‐blotting using the indicated antibodies. (b) Nuclear lysates were analysed by immuno‐blotting using antibodies against RELB, P65, REL and H2B Histone. One representative experiment of three is shown. Relative protein expression levels were quantified by image studio lite software. (c) Quantification of phospho‐p65 levels in total cell lysates obtained from thymocytes treated as described for (a). n = 3.

Article Snippet: Membranes were probed with antibodies against pp65, I κ B α , pJNK, pERK1/2, pp38, RELB (Cell Signaling, Danvers, MA) or p38, p65, cREL, H2B Histone (Santa Cruz Biotechologies, Dallas, TX) and visualized using enhanced chemiluminescence (Pierce Biotechnology, Rockford, IL) for detection.

Techniques: Activation Assay, Isolation, Expressing, Software

Journal: The Journal of Neuroscience

Article Title: A Novel Activator of CBP/p300 Acetyltransferases Promotes Neurogenesis and Extends Memory Duration in Adult Mice

doi: 10.1523/JNEUROSCI.5772-12.2013

Figure Lengend Snippet: : CSP-TTK21 is located in the brain after a single intraperitoneal (i.p.) injection. A, Localization of CSP-TTK21 in different organs of mice 1 d after an i.p. injection. Mice (n = 5) were injected with 20 mg/kg CSP-TTK21 and killed 24 h later. Brain, liver, lungs, kidney, and spleen were isolated and processed for histochemistry. CSP-TTK21 was detected by excitation at 514 nm. Nucleus of the cells in various tissues was stained with Hoechst. Histograms represent the quantification of the percentage of cells containing intranuclear CSP-TTK21. After 1 d, CSP-TTK21 molecules are mainly found in the brain, liver, and spleen. B, C, Time-dependent presence of CSP-TTK21 in mice liver (B) and spleen (C) after a single i.p. injection. Same as in A, except that 3 groups of mice were used (n = 3) and killed at different days after injection as noted (1, 3, and 7 d). CSP-TTK21 molecules levels are largely reduced 3 d after injection and almost absent after 7 d. D, E, Time-dependent presence of CSP-TTK21 in mice brain after a single i.p. injection. Five groups of mice (n = 3) that were used (n = 3) and killed at different days after injection as noted (1, 3, 7, 14, and 21 d). Typical photographs for days 1 and 3 are shown (D). Magnified image of the nucleus from days 1 and 3 showing localization of CSP-TTK21 in the nuclei (inset of the respective figures). Scale bar, 10 μm. E, Quantification of the percentage of cells containing intranuclear CSP-TTK21. Note that large amounts of CSP-TTK21 molecules are located in the nuclei of brain cells at 3 d after injection and that the molecules are rapidly cleared out of cells. F, G, Three days after i.p. injection of either CSP alone or CSP-TTK21 (20 mg/kg body weight), different organs or brain subregions were dissected out and Western blots were performed on total protein extracts with acetylated H2B or H3 and total H2B. Quantification of acetylated histone levels is shown relative to the total amount of the H2B (n = 4–6). Student's t test, *p < 0.05 and **p < 0.01 compared with CSP control. #Close to significance. Cx indicates cortex; d, dorsal; Hippo, hippocampus; v, ventral; p, para.

Article Snippet: Western blots were performed as described previously ( Bousiges et al., 2010 ) with antibodies against acetyl-histone H2B (#07-373; Millipore), acetylated-histone H2BK5 (#07-382; Millipore), H3 histone (#ab1791; Abcam), acetyl histone H3 lys 14 (#1921; Epitomics), acetyl Histone H3 (#06-599; Millipore), and H2B histone (#H2-28; Euromedex); pro-BDNF (#AB9612; Millipore), GFAP (#MAB360; Millipore), NeuN (#ABN78; Millipore), and β-actin (#ab16039; Abcam).

Techniques: Injection, Isolation, Staining, Western Blot

Journal: The Journal of Neuroscience

Article Title: A Novel Activator of CBP/p300 Acetyltransferases Promotes Neurogenesis and Extends Memory Duration in Adult Mice

doi: 10.1523/JNEUROSCI.5772-12.2013

Figure Lengend Snippet: CSP-TTK21 induces histone acetylation on several residues of H2B and H3 in the mouse dorsal hippocampus. Mice were injected with either CSP alone or CSP-TTK21 (20 mg/kg each) and killed 3 d later. A, Immunohistochemistry was performed on 20-μm-thick cryosections with an anti-acetylated-H2BK5 antibody. Acetylation was increased in all hippocampal areas. Scale bar, 200 μm. A focus on the dentate gyrus is shown below. Scale bar, 100 μm. B, C, Immunofluorescent labeling of mouse dorsal hippocampus using antibodies against tetra-acetylated H2B (B) and acetylated-H3K14 (C). Scale bar, 100 μm.

Article Snippet: Western blots were performed as described previously ( Bousiges et al., 2010 ) with antibodies against acetyl-histone H2B (#07-373; Millipore), acetylated-histone H2BK5 (#07-382; Millipore), H3 histone (#ab1791; Abcam), acetyl histone H3 lys 14 (#1921; Epitomics), acetyl Histone H3 (#06-599; Millipore), and H2B histone (#H2-28; Euromedex); pro-BDNF (#AB9612; Millipore), GFAP (#MAB360; Millipore), NeuN (#ABN78; Millipore), and β-actin (#ab16039; Abcam).

Techniques: Injection, Immunohistochemistry, Labeling