Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: Tissue-Type Plasminogen Activator Regulates the Neuronal Uptake of Glucose in the Ischemic Brain

doi: 10.1523/JNEUROSCI.1241-12.2012

Figure Lengend Snippet: A. Experimental design used to study the effect of tPA on neuronal survival. Letters denote time of treatment with tPA after exposure to oxygen-glucose deprivation (OGD) conditions. B – D. Mean cell survival (panels B & C) and release of LDH into the culture media (panel D) in Wt cerebral cortical neurons treated with 5 nM of proteolytically active (panels B & D) or inactive tPA (itPA; panels C & D), or 10 nM of plasmin (panel C), 5, or 30, or 60, or 120, or 180, or 360 minutes after exposure to 55 minutes of OGD conditions. n = 20 in B and 15 in C and D. * in B: p < 0.05 compared to neurons exposed to OGD conditions without subsequent treatment with tPA. * in C: p < 0.05 compared to neurons left untreated after exposure to OGD conditions. Ns: non-significant. * in D: p < 0.05 compared to cells exposed to OGD conditions without subsequent treatment. Lines denote SD. E. Mean cell survival in Wt cerebral cortical neurons exposed to 55 minutes of OGD conditions and treated 1 hour later with 5 nM of tPA, alone or in combination with either 10 µM of MK-801 or 60 nM of the receptor associated protein (RAP), or 100 nM of the tyrosine kinase receptor B (TrkB) inhibitor K-252a. n = 10. * p < 0.05 compared to neurons treated with tPA alone, or with a combination of tPA and K-252a. Lines denote SD. F. Mean volume of the ischemic lesion in Wt and Plg−/− mice treated one hour after tMCAO with saline solution (white bars) or rtPA 1 – 9 mg/Kg/IV (gray bars). n = 10 per group. * p < 0.05 compared to Wt mice treated with saline solution. ** p < 0.05 compared to mice treated with 1 mg/Kg/IV of rtPA. *** p < 0.05 compared to Plg−/− mice treated with saline solution. Bars depict mean volume of the ischemic lesion in mm3. Lines denote SD.

Article Snippet: Other reagents were human recombinant tissue-type plasminogen activator (Genentech Inc.), the phosphoinositide (PI) 3-kinase/Akt inhibitor Wortmannin, methanol, methyl salicylate and triphenyltetrazolium chloride (Sigma Aldrich), the 3-(4,5- Dimethylthiazol -2-yl)-2,5-di phenyl tetrazolium bromide (MTT) assay (ATCC), the LDH release assay (Roche), the Receptor-Associated Protein (RAP; kindly provided by Dr. Dudley K. Strickland, University of Maryland), the NMDAR antagonist MK-801 (Tocris Bioscience), rapamycin and the TrkB inhibitor K-252a (Calbiochem), HIF-1α shRNA, scramble shRNA lentiviral particles, anti-GLUT3 antibodies and TRITC-conjugated donkey anti-goat IgG (Santa Cruz Biotechnology), anti-HIF-1α antibodies (Abcam), antibodies against the p70S6 kinase (p70 S6K ) phosphorylated at Thr389 (Cell Signaling), heparin sodium (Abraxis Pharmaceutical Products), blue latex (Connecticut Valley Biological Supply), ApopTag Plus Fluorescein in Situ Apoptosis Detection Kit (Chemicon International), 4'-6-Diamidino-2-phenylindole (DAPI; Invitrogen), triphenyltetrazolium chloride (TTC; Sigma-Aldrich), 2- N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG; Molecular Probes), and 18-Fluorodeoxyglucose (PETNET).

Techniques: Saline

Journal: The Journal of Cell Biology

Article Title: Rapid Retrograde Tyrosine Phosphorylation of trkA and Other Proteins in Rat Sympathetic Neurons in Compartmented Cultures

doi:

Figure Lengend Snippet: Protein tyrosine phosphorylation in response to different distributions of NGF. Compartmented cultures of rat sympathetic neurons were grown for 2 wk in 10 ng/ml NGF in all compartments. Cultures were treated for 10 min with either: ( a ) 10 ng/ml NGF applied to all compartments; ( b , d , and e ) 10 ng/ml NGF applied to cell bodies/proximal axons and 200 ng/ml NGF applied to distal neurites; or ( c ) 200 ng/ml NGF applied to all compartments. In center compartments ( d ) and in all compartments ( e ), 500 nM K-252a ( K2 ) was supplied, starting 30 min before the NGF treatments. Cell extracts were collected from the cell body/proximal neurite compartments ( CB ) and the distal neurite compartments ( N ). To ensure comparability between treatments, all cultures used were sister cultures, and each group contained the extracts pooled from three cultures. The extracts were analyzed by immunoblotting with anti-phosphotyrosine (4G10) antibody. ( Arrows ) Tyrosine phosphorylation of proteins produced by distally applied NGF. ( Asterisks ) Tyrosine-phosphorylated proteins found only in cell bodies/proximal axons. The position of trk migration is indicated. Molecular mass markers are indicated on the left.

Article Snippet: K-252a (Kamiya Biomedical Co., Thousand Oaks, CA) was prepared as a 2 mM stock in DMSO and stored at 4°C.

Techniques: Western Blot, Produced, Migration

Journal:

Article Title: Okadaic Acid Mimics Nitrogen-Stimulated Transcription of the NADH-Glutamate Synthase Gene in Rice Cell Cultures 1

doi:

Figure Lengend Snippet: Effects of protein kinase inhibitors on the induction of NADH-GOGAT mRNA by NH4+ and OKA. Suspension-cultured rice cells were pretreated with 1 μm staurosporine (STA) or 1 μm K-252a for 1 h, and then treated with 20 mm NH4Cl or 1 μm OKA for 6 or 12 h, respectively. Total RNA was isolated and subjected to RNA gel-blot analysis using the digoxigenin-labeled cDNA probes for NADH-GOGAT or tubulin. The ethidium-bromide-stained ribosomal bands are shown as a loading control.

Article Snippet: OKA and calyculin A were from Wako Pure Chemical Industries (Osaka), 1-norokadaone, staurosporine, and K-252a from Nacalai Tesque (Kyoto), and all were dissolved in DMSO at 1 m m as a stock solution.

Techniques: Cell Culture, Isolation, Western Blot, Labeling, Staining