Journal: PLoS ONE

Article Title: Activation of JNK Triggers Release of Brd4 from Mitotic Chromosomes and Mediates Protection from Drug-Induced Mitotic Stress

doi: 10.1371/journal.pone.0034719

Figure Lengend Snippet: A. P19 cells transfected with vectors for GFP alone or full length GFP-Brd4 (GFP-FL-Brd4) were treated with nocodazole (50 ng/ml) for 4 h, and GFP signals were viewed by confocal and differential interference contrast (DIC) microscopy. B. Differential salt extraction. Mitotic cells treated with nocodazole, paclitaxel or colcemid (100 ng/ml each) for 4 h were extracted with indicated concentrations of KCl. Twenty µg of extracts were immunoblotted with antibody for Brd4 or TFIIB. In the right panel, total Brd4 and TFIIB extracted at 400 mM KCl were detected as above. C. Cells were treated with indicated concentrations of nocodazole for 4 h, and mitotic cells were immunostained for endogenous Brd4 and α-tubulin. DNA was stained by Hoechst33324. D. Mitotic indices of nocodazole treated cells. Cells in prometaphase or anaphase/telophase were microscopically inspected. Approximately 200 cells were examined for each data point. E. Cells were treated with 100 µM of monastrol or 50 µM of blebbistatin for 4 h and localization of Brd4 was examined by immunostaining as in .

Article Snippet: Extracted proteins were resolved on 4–12% SDS PAGE (Invitrogen), transferred to Immobilon membrane (Millipore) and immunoblotted with anti-Brd4 antibody or anti-TFIIB antibody (Santa Cruz).

Techniques: Transfection, Microscopy, Staining, Immunostaining

Journal: bioRxiv

Article Title: Optogenetic clustering and membrane translocation of the BcLOV4 photoreceptor

doi: 10.1101/2022.12.12.520131

Figure Lengend Snippet: A) The intracellular domains of ErbB1-4 were fused to the C-terminus of BcLOV-mCherry. B) Membrane translocation of BcLOV-ErbB1-4 in response to blue light. Scale bar = 20 μm. See also Supplementary Movie 3 . C,D) Erk activation dynamics downstream of BcLOV-ErbB1-4 in response to ON and OFF steps of blue light. See also . Data represent mean ± SEM of two replicates, with each replicate representing the mean of ~500-2400 cells. E) Membrane ruffling (black arrows) downstream of stimulation of BcLOV-ErbB1-4, indicative of RTK stimulation. Ruffling is strongest for ErbB1 and ErbB2, less for ErbB4, and absent for ErbB3 activation. Scale bar = 20 μm. See also Supplementary Movie 4 . F) Kinase activity suppresses BcLOV-EGFR membrane translocation. Translocation was observed under light stimulation in the presence or absence of 1 μM EGFR inhibitor erlotinib (EGFRi). EGFRi promoted stronger translocation. Scale bar = 20 μm. Quantification (right) shows ratios of mean membrane and cytoplasmic fluorescence of 25 single cells.

Article Snippet: ErbB4 was sourced from pDONR223-ERBB4, which was a gift from William Hahn & David Root (Addgene plasmid # 23875; http://n2t.net/addgene:23875 ; RRID:Addgene_23875). iLID, sspB, and SOS cat were sourced from previously described constructs .

Techniques: Translocation Assay, Activation Assay, Activity Assay, Fluorescence

Journal: BMC Molecular Biology

Article Title: Association of ATRX with pericentric heterochromatin and the Y chromosome of neonatal mouse spermatogonia

doi: 10.1186/1471-2199-9-29

Figure Lengend Snippet: Specific association of ATRX and H3K9 me3 with lack of H3K4 me2 at repetitive DNA sequences on the Y chromosome . A) Metaphase spreads of peripheral lymphocytes (top panel) and primary fibroblasts (lower panel). Thin arrows indicate the characteristic association of ATRX (green) at pericentric heterochromatin in the autosomes of both cell lineages. ATRX consistently labels the Y chromosome (red) in mouse embryonic fibroblasts (bold arrow) but is undetectable in the Y chromosome of peripheral lymphocytes. Scale bars = 10 μm. B) Chromatin immunoprecipitation (ChIP) analysis of histone modifications associated with a Y-chromosome specific repetitive sequence. ChIP assays were performed on male embryonic fibroblasts (upper panel) and peripheral lymphocytes (lower panel) using antibodies directed against ATRX, H3K4 me2 and H3K9 me3 . Representative gel images are depicted above the corresponding graphs. A rabbit IgG (lanes 3 and 4) and an anti-TFIIB antibody (lanes 11 and 12) served as negative and positive control, respectively. In primary fibroblasts, the specific association of H3K9 me3 (lanes 9 and 10) and ATRX (lanes 7 and 8) with repetitive sequences on the Y chromosome results in a significant enrichment of co-precipitated DNA fragments corresponding to the Y chromosome-specific repeat (Y666) compared to a non-specific IgG negative control (lanes 3 and 4) and the H3K4 me2 antibody (lanes 5 and 6). However, although H3K9 me3 is also enriched at these sequences in peripheral lymphocytes (lower panel), ATRX associations are not significantly different from samples precipitated with the negative control (IgG) or H3K4 me2 . PCR amplification was conducted using increasing amounts of precipitated template (input) to ensure that the PCR reaction was within the linear range. Error bars represent the SEM of three independent experiments and different superscripts indicate significant differences (p < 0.05).

Article Snippet: Experiments were carried out in triplicates using an antibody for the transcription factor TFIIB (Santa Cruz) as a positive control, as well as antibodies for the ATRX protein (Santa Cruz), anti-H3K4 me2 (Upstate) and an anti-H3K9 me3 (Abcam) antibody.

Techniques: Chromatin Immunoprecipitation, Sequencing, Positive Control, Negative Control, Amplification