Journal: PLoS ONE

Article Title: Transcription factor-induced activation of cardiac gene expression in human c-kit+ cardiac progenitor cells

doi: 10.1371/journal.pone.0174242

Figure Lengend Snippet: A, Untranduced or mCherry virus-transduced CPCs were imaged at 4 days post-transduction. Fluorescence of mCherry protein is visible in red. DAPI staining of nuclei was pseudo-colored in green. B, Cells transduced with virus expressing mCherry (control), 3xFLAG-tagged Gata4, MEF2C, TBX5 or BAF60C, or V5-tagged NKX2.5 were assayed by Western blot using antibody against each TF. C, Cells transduced with virus expressing 3xFLAG-tagged Gata4, MEF2C, TBX5 or BAF60C, or V5-tagged NKX2.5 were stained for the indicated epitope (i.e., FLAG or V5) which is shown in monochrome. DAPI images are shown in lower panels. Note that exogenous, epitope-tagged transcription factors localize to the nucleus as expected.

Article Snippet: KDR/VEFGR2 (mouse monoclonal; ab9530; Abcam); α-SMA (mouse monoclonal; A5228; Sigma); troponin T (mouse monoclonal; clone 13–11; Thermo Fisher); α-sarcomeric actinin (mouse monoclonal; A7811; Sigma); FLAG tag (mouse monoclonal; F-tag-01; Applied Biological Materials); Thy1/CD90 (Mouse monoclonal; clone 5E10; BD Pharmingen); SM-MHC (rabbit polyclonal; Abcam); ANP (mouse monoclonal; clone 23/1; Santa Cruz); BNP (mouse monoclonal; clone 50E1; Thermo Fisher); c-kit (rabbit monoclonal; clone YR145; Epitomics); α-tubulin (mouse monoclonal; clone DM1A; Sigma); V5 tag (mouse monoclonal; clone E10; Applied Biological Materials).

Techniques: Virus, Transduction, Fluorescence, Staining, Expressing, Control, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: TRIM24 protein promotes and TRIM32 protein inhibits cardiomyocyte hypertrophy via regulation of dysbindin protein levels

doi: 10.1074/jbc.M116.752543

Figure Lengend Snippet: TRIM24 and TRIM32 interact with cardiac dysbindin. HEK293A cells were co-transfected with V5-tagged dysbindin and FLAG-tagged TRIM24 or TRIM32. Empty vectors transfected with either dysbindin, TRIM24, or TRIM32 were used as respective controls. Immunoprecipitation was performed using anti-V5 or FLAG tag cross-linked magnetic beads. Precipitated proteins were immunoblotted with respective antibodies. Dysbindin was found to be co-precipitated with TRIM24 (A) and vice versa (B). Similarly, TRIM32 and dysbindin pulled down each other (C and D, respectively), although no interaction was seen in control IP (lane 1 in each blot), confirming interaction between dysbindin and TRIM24/TRIM32. We also performed co-IP by overexpressing HA-tagged dysbindin in NRVCMs and precipitated using anti-HA tag cross-linked magnetic beads. Precipitated proteins were immunoblotted with TRIM24 (E) and TRIM32 (F) antiserum. and the interaction between dysbindin and TRIM24 or TRIM32 was further validated. Vertical black lines in the blots indicate that the intervening lanes have been spliced out. G, confocal micrographs representing the co-immunostaining of endogenous dysbindin with either TRIM24 (upper lane) or TRIM32 (lower lane). H, co-immunostaining of native dysbindin, TRIM24, or TRIM32 with α-actinin. Nuclei were stained with DAPI, and images were captured with a Zeiss LSM800 laser-scanning microscope. Scatter plots show the analysis of co-localization as described under “Experimental procedures.” White rectangles represent cropped areas for detailed re-acquisition. MCC = Mander's co-localization coefficient. Scale bar (shown with a white line), 20 μm. IP, immunoprecipitation; WB, Western blotting; Dys, dysbindin.

Article Snippet: Antibodies used for various experiments in this study were as follows: actin, goat polyclonal (1:3000; Santa Cruz Biotechnology), α-actinin, mouse monoclonal (1:200; Sigma); α-actinin, rabbit polyclonal (1:400; Abcam); α-tubulin, mouse monoclonal (1:8,000; Sigma); caspase-3, rabbit polyclonal (1:1,000; Cell Signaling Technology); cleaved caspase-3, rabbit monoclonal (1:400; Cell Signaling Technology); caspase-7, rabbit polyclonal (1:1,000; Cell Signaling Technology); dysbindin, mouse monoclonal (1:500, Santa Cruz Biotechnology); FLAG tag, mouse monoclonal (1:500; Sigma); GAPDH (1:20,000; Sigma); p53, mouse monoclonal (1:1,000, Novus Biologicals); TRIM24, rabbit polyclonal (1:1,000; Proteintech); TRIM32, rabbit polyclonal (1:500, Sigma); ubiquitin, mouse monoclonal (1:1,000; Millipore Upstate); V5 tag, mouse monoclonal (1:500; Biozol); and XIAP, rabbit polyclonal (1:1,000; Cell Signaling Technology).

Techniques: Transfection, Immunoprecipitation, FLAG-tag, Magnetic Beads, Co-Immunoprecipitation Assay, Immunostaining, Staining, Laser-Scanning Microscopy, Western Blot

Journal: Molecular cell

Article Title: FUS Regulates Activity of MicroRNA-mediated Gene Silencing

doi: 10.1016/j.molcel.2018.02.001

Figure Lengend Snippet: Key Resources Table

Article Snippet: V5 mouse monoclonal , , , LifeTech , 460705.

Techniques: Negative Control, Recombinant, Reporter Assay, Generated, Binding Assay, Mutagenesis, Software, Microarray, Expressing