Journal: Journal of Biological Chemistry

Article Title: Real Time Analysis of STAT3 Nucleocytoplasmic Shuttling

doi: 10.1074/jbc.m312530200

Figure Lengend Snippet: FIG. 1. STAT3-CY fusion proteins. A, domain structures of fluorescent STAT3 fusion proteins used in this study. Numbers refer to the respective amino acids. B, COS7 cells were cotransfected with the indicated fluorescent STAT3 constructs and IL-5R/-gp130 chimeras (38). 48 h after transfection cells were stimulated with 20 ng/ml IL-5 for 15 min. Cellular lysates were prepared, and fusion proteins were precipitated (IP) with a GFP antibody. Precipitates were analyzed by Western blotting using GFP, N-terminal STAT3, and P-STAT3 antibodies as indicated. C, induction of the 2-macroglobulin promoter by endogenous STAT3 (mock-transfected) or cotransfected wild type STAT3, STAT3-YFP, or STAT3-CY measured in a reporter gene assay. Cells were stimulated with 20 ng/ml IL-6 for 16 h or left without stimulation.

Article Snippet: Antibodies against STAT3 (BD Biosciences), phosphorylated STAT3 (Sigma), and GFP (Rockland Immunochemicals, Gilbertsville, PA) were used for immunoprecipitation (3 l/ml lysate) and for Western blot analysis (1:1,000 dilution).

Techniques: Construct, Transfection, Western Blot, Reporter Gene Assay

Journal: International Journal of Medical Sciences

Article Title: Parvovirus B19 Nonstructural Protein-Induced Damage of Cellular DNA and Resultant Apoptosis

doi:

Figure Lengend Snippet: PARP is active and necessary for efficient apoptosis in NS1-transfected cells. A. Immunoprecipitated GFP/NS1 protein was poly(ADP ribose)ylated, as shown by a band at 100 kd on the blot probed with anti-poly ADP ribose (PAR, left blot) antibodies. The blots were stripped and reprobed with anti-GFP (right), showing that PAR colocalizes with the GFP/NS1 fusion protein. GFP alone is not ribosylated, as evidenced by the lack of a PAR band corresponding to GFP at 37 kD. Blots shown are representative of three independent experiments. B. PARP activity is necessary for optimal NS1-induced apoptosis. Addition of the PARP inhibitor 5-aminoisoquinolinone (5AIQ) to GFP/NS1 expressing HepG2 cells reduced apoptosis by 57% ( p <0.003). Addition of 5-aminoisoquinolinone had no effect on the GFP transfected cells. N=3, error bars indicate the range of values.

Article Snippet: 25 μl of anti-GFP polyclonal antibody (Rockland, Gilbertsville, PA) were added and the mixture was allowed to bind for 14 hours at 4 o C in an end-over end rotator.

Techniques: Transfection, Immunoprecipitation, Activity Assay, Expressing

Journal: eLife

Article Title: A vibrissa pathway that activates the limbic system

doi: 10.7554/eLife.72096

Figure Lengend Snippet: ( A ) Viral method used for labeling paralemniscal projections. ( B ) Labeling of interpolaris cells after injection of G-pseudotyped Lenti-Cre virus in Po thalamus, and a Cre-dependent AAV that expresses GFP in the vibrissa-responsive sector of SpVIr. Horizontal section. ( C ) Anterograde labeling of terminal fields in Po thalamus and zona incerta. Sagittal section. ( D ) Anterograde labeling in the KF/PBc. Horizontal section. ( E ) Anterograde labeling in the ITr. Horizontal section. ( F ) Anterograde labeling in the dorsal sector of the facial nucleus. Horizontal section. ( G ) Anterograde labeling in the MdD and cervical cord. Horizontal section. ( H ) Population peristimulus time histogram of spike discharges evoked in KF (22 cells) by air puff deflection of the vibrissae in the anesthetized rat. A representative response is shown in the insert. ( I ) Example of a vibrissa responsive KF cell labeled by juxtacellular delivery of Neurobiotin. Horizontal section. ( J ) Location of eight juxtacellularly labeled KF cells. Horizontal brainstem sections in ( I ) and ( J ) were counterstained for cytochrome oxidase. Horizontal section. ( K ) Spectral coherence of spontaneous discharges of KF cells with the respiratory cycle at the respiratory frequency; 1–3 Hz. Note that, in contrast with the respiratory units (blue dots), spontaneous discharges of vibrissa-responsive cells (red triangles) display low coherence with respiration. ( L ) Population peristimulus time histogram of spike discharges evoked in MdD (33 cells) by air puff deflection of the vibrissae. ( M ) Recording site in the MdD labeled by an iontophoretic injection of Chicago Sky Blue. This coronal section was counterstained for cytochrome oxidase and a negative image was generated. Coronal section. See for additional anatomical data. Abbreviations for all anatomy: 5n, root of the trigeminal motor nucleus; 5N, trigeminal motor nucleus; 5t, trigeminal tract; 7n, facial nerve tract; 7N, facial nucleus; Amb, ambiguus nucleus; APT, anterior pretectal nucleus; BSTL, bed nucleus of the stria terminalis; CeA, central amygdala; Cerv Cord, cervical cord; CM/PC, central medial/paracentral thalamic nuclei; CPu, caudate putamen; DR, dorsal raphe; EW, Edinger-Westphal; Hab, habenula; IML, intermedio-lateral column of the spinal cord; ITr, intertrigeminal region; KF, Kölliker-Fuse nucleus; KF/PBc, Kölliker-Fuse/parabrachial complex; mcp, middle cerebellar peduncle; MdD, dorsal part of the medullary reticular formation; MdV, ventral part of the medullary reticular formation; mt, mammillothalamic tract; NA, nucleus ammbiguus; NTS, nucleus of the solitary tract; opt, optic tract; PAG, periaqueductal gray; PB, parabrachial nuclei; PC, paracentral thalamic nucleus; PCRt, parvicellular reticular formation; PLH, posterior lateral hypothalamus; Po, posterior nuclear group of the thalamus; PrV, principal trigeminal nucleus; RN, red nucleus; Rt, reticular thalamic nucleus; s5, sensory root of the trigeminal nerve; SC, superior colliculus; scp, superior cerebellar peduncle; SpVC, caudalis division of the spinal trigeminal complex; Sol, nucleus of the solitary tract; SpVIc, caudal sector of the interpolaris trigeminal nucleus; SpVIr, rostral division of the interpolaris nucleus; TG, trigeminal ganglion; VLL, ventral nucleus of the lateral lemniscus; VPL, ventral posterolateral thalamic nucleus; VPM, ventral posterior medial nucleus; VPCc, parvicellular sector of the ventral posteromedial thalamic nucleus; VPPc, parvocellular part of the ventral posterior thalamic nucleus; VRG, ventral respiratory group; vsc, ventral spinocerebellar tract; ZIv, ventral division of zona incerta.

Article Snippet: For brightfield microscopy, sections were first counterstained for cytochrome oxidase (MilliporeSigma), and then immunoreacted with a rabbit anti-GFP antibody (1:1000; Novus Biological), a biotinylated horse anti-rabbit IgG (1:200; Vector Labs), the avidin/biotin complex (Vectastain ABC Kit; Vector Labs), and the SG peroxidase substrate (ImmPACT SG Substrate; Vector Labs).

Techniques: Labeling, Injection, Virus, Generated

Journal: eLife

Article Title: A vibrissa pathway that activates the limbic system

doi: 10.7554/eLife.72096

Figure Lengend Snippet:

Article Snippet: For brightfield microscopy, sections were first counterstained for cytochrome oxidase (MilliporeSigma), and then immunoreacted with a rabbit anti-GFP antibody (1:1000; Novus Biological), a biotinylated horse anti-rabbit IgG (1:200; Vector Labs), the avidin/biotin complex (Vectastain ABC Kit; Vector Labs), and the SG peroxidase substrate (ImmPACT SG Substrate; Vector Labs).

Techniques: Plasmid Preparation, Recombinant, Virus, Software