Journal: Cells

Article Title: Annexin A2 Egress during Calcium-Regulated Exocytosis in Neuroendocrine Cells

doi: 10.3390/cells9092059

Figure Lengend Snippet: Tissue plasminogen activator (t-PA) is present on the outer leaflet of the plasma membrane of stimulated chromaffin cells. ( a ) Dual labeling of cell surface t-PA and exocytotic sites. Cells were stimulated with nicotine 20 µM in the presence of anti-t-PA and anti-DBH antibodies. Cells were then fixed and incubated with secondary antibodies coupled to Alexa Fluor ® 488 and Alexa Fluor ® 551, respectively. Confocal images were recorded in the same optical section and with the same parameters of lasers and photomultipliers. Scale bar: 10 µM. ( b ) The t-PA labeling on the surface of chromaffin cells in the resting condition (R), stimulated with 20 µM nicotine without (S) or after EGTA wash (S + EGTA). Anti-t-PA antibodies were revealed with Alexa Fluor ® 488-conjugated anti-rabbit antibodies and F-actin with TRITC-phalloidin to visualize the cell shape. Confocal images were recorded in the same optical section by a dual exposure procedure. Scale bar: 10 µM. ( c ) Semi-quantitative analysis of t-PA labeling on the cell surface of chromaffin cells in the resting condition (R), stimulated with 20 µM nicotine (S) without (Control) or after EGTA wash (S + EGTA). The t-PA labeling is expressed in arbitrary units. Statistical significance for medians was determined using a Mann-Whitney test. Dotted lines indicate the mean and asterisks statistical significance (*** = p < 0.001, ** = p < 0.01). Three experiments were done on independent cell cultures and pooled ( n = 36 and 65 control cells, 40 and 75 EGTA-treated cells for resting and stimulated conditions, respectively). ( d ) Lysate, secreted material and EGTA eluate from chromaffin cells in the resting condition (R) or stimulated with nicotine 20 µM (S) were analyzed by western blot and revealed with anti-t-PA and anti-AnxA2 antibodies. Data correspond to a typical experiment representative of three independent experiments.

Article Snippet: Rabbit polyclonal anti-mouse tissue plasminogen activator (t-PA) antibodies were from Molecular Innovations (Novi, MI, USA).

Techniques: Labeling, Incubation, MANN-WHITNEY, Western Blot

Journal: Cells

Article Title: Annexin A2 Egress during Calcium-Regulated Exocytosis in Neuroendocrine Cells

doi: 10.3390/cells9092059

Figure Lengend Snippet: Membrane topography of AnxA2, t-PA and exocytotic sites after immunogold labeling of the outer face of the plasma membrane sheets prepared from stimulated chromaffin cells. ( a ) Plasma membrane sheets were prepared from bovine chromaffin cells stimulated by nicotine for 5 min. To label DBH, AnxA2 and t-PA exposed at the surface of cells undergoing exocytosis, anti-DBH, anti-AnxA2 and anti-t-PA antibodies were added during stimulation. Membrane sheets were labeled with anti-mouse antibodies coupled to 10 nm gold particles to detect DBH antibodies revealing exocytotic sites (red circle) and rabbit antibodies coupled to 15 nm gold particles to label AnxA2 or t-PA (green circle). ( b ) The histogram represents the relative distribution of 15 nm gold particles as a function of their distance from the granule membrane once inserted in the plasma membrane (blue line). The distance was measured and the number of particles was counted manually with Photoshop. Three experiments were done on independent cell cultures ( c ). Double staining experiment for t-PA (10 nm gold particles) and AnxA2 (15 nm gold particles) were performed with the same protocol. Scale bar: 100 nm. ( d ) The histogram represents the relative distribution of 10 nm gold particles (t-PA) as a function of their distance from 15 nm gold particles (AnxA2). The distance was measured and the number of particles was counted manually with Photoshop. Two experiments were done on two independent cell cultures.

Article Snippet: Rabbit polyclonal anti-mouse tissue plasminogen activator (t-PA) antibodies were from Molecular Innovations (Novi, MI, USA).

Techniques: Labeling, Double Staining

Journal: Scientific Reports

Article Title: Tissue Plasminogen Activator Neurotoxicity is Neutralized by Recombinant ADAMTS 13

doi: 10.1038/srep25971

Figure Lengend Snippet: ( A ) Representative immunoblots and quantitative analysis of the calpain- (145/150 kDa) and caspase-specific fodrin cleaved products (120 kDa) in sham-operated mice, and mice treated with vehicle, tPA, tPA in combination with rADAMTS 13, and rADAMTS 13 24 hours after MCA occlusion. The gels have been run under the same experimental conditions. ( B ) Six fields selected for immunohistochemical quantification of LC3, TUNEL, and FJB-labeled cells in the peri-infarct area in Nissl-stained coronal sections in all the groups. ( C ) Representative photomicrographs of TUNEL staining and quantification of TUNEL-positive cells in the ischemic brain of mice treated with vehicle, tPA, rADAMTS 13 in combination with tPA, and rADAMTS 13 24 hours after MCA occlusion. ( D ) Representative photomicrographs of Fluoro-Jade B (FJB) staining and quantification of FJB-positive cells in the ischemic brain of mice treated with vehicle, tPA, rADAMTS 13 in combination with tPA, and rADAMTS 13 24 hours after MCA occlusion. ( E ) Representative immunoblots of LC3 and quantification of the LC3-II/LC3-I ratio in sham-operated mice, and mice treated with vehicle, tPA, tPA in combination with rADAMTS 13, and rADAMTS 13 24 hours after MCA occlusion. The gels have been run under the same experimental conditions. ( F ) Representative photomicrographs of LC3 staining and quantification of LC3-positive cells in the ischemic brain of mice treated with vehicle, tPA, rADAMTS 13 in combination with tPA, and rADAMTS 13 24 hours after MCA occlusion. Bar = 30 μm. Values are mean ± SD (n = 5 per group). *P < 0.05.

Article Snippet: The primary antibodies were rabbit anti-LC3 (Light Chain 3, 4108), rabbit anti-pERK1/2 (phosphorylation of extracellular signal-regulated kinase1/2, 9101), rabbit anti-pJNK (c-Jun N-terminal kinases, 9251), rabbit anti-p-p38 (9211), rabbit anti-NR1 (N-methyl-D-aspartate receptor, 4204), rabbit anti-NR2A (4205), rabbit anti-NR2B (4207), rabbit anti-β-actin (4967) (all from Cell Signaling Technology, Beverly, MA, USA), mouse anti-α-fodrin (AA6, Enzo Life Sciences, Inc. Farmingdale, NY, USA), rabbit anti-tPA (ASHTPA-GF-HT, Molecular Innovations Inc., Novi, MI, USA).

Techniques: Western Blot, Immunohistochemical staining, TUNEL Assay, Labeling, Staining