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: Journal of Cerebral Blood Flow & Metabolism

Article Title: Tissue-type plasminogen activator mediates neuronal detection and adaptation to metabolic stress

doi: 10.1038/jcbfm.2013.124

Figure Lengend Snippet: Glucose deprivation (GD) induces the release of tissue-type plasminogen activator (tPA) from the presynaptic terminal of cerebral cortical neurons. (A) Representative micrograph of wild-type (Wt) cerebral cortical neurons maintained during 5 minutes under 25 mmol/L (+G) or 0 mmol/L (−G) of glucose and stained with antibodies against the postsynaptic dendritic marker anti-microtubule associated protein (MAP-2) (blue) and tPA (red). Green corresponds to DNA (Hoechst). Magnification × 20 in (a, e) and × 60 in (b–d) and (f–h). (B) Representative micrograph of Wt cerebral cortical neurons maintained during 5 minutes under GD and stained with antibodies against MAP-2 (blue), tPA (red), and synaptophysin (green). Arrows denote the presence of tPA-containing synaptophysin-positive presynaptic vesicles in direct contact with the postsynaptic, MAP-2-positive, dendrite. Magnification × 60. (C) Mean concentration of tPA in the culture medium of Wt cerebral cortical neurons exposed during 0 to 5 minutes to either oxygen deprivation (OD, black circles), or GD (black triangles), or oxygen and glucose deprivation (OGD; black squares), or kept under normal glucose and oxygen concentrations after medium change (baseline; white diamonds). Lines denote s.d. n=10 per condition; *P<0.05 compared with GD and OD; ^P<0.05 compared with OD and OGD; §P<0.05 compared with sister cultures maintained under physiologic conditions and with cultures exposed to either GD or OGD conditions.

Article Snippet: Recombinant murine tPA, proteolytically inactive tPA (itPA) with an alanine for serine substitution at the active site Ser481 (S481A), human Lys plasmin, an ELISA kit that detects active tPA, and sheep anti-tPA antibodies (Cat # SASMTPA) were acquired from Molecular Innovations (Novi, MI, USA).

Techniques: Staining, Marker, Concentration Assay

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Tissue-type plasminogen activator mediates neuronal detection and adaptation to metabolic stress

doi: 10.1038/jcbfm.2013.124

Figure Lengend Snippet: Tissue-type plasminogen activator (tPA) induces adenosine monophosphate-activated protein kinase (AMPK) activation in the postsynaptic terminal via N-methyl-D-aspartate receptors (NMDARs) activation. (A) Representative western blot analysis of pAMPK expression in non-ischemic wild-type (Wt) cerebral cortical synaptoneurosomes after 5 minutes of incubation with 5 nmol/L of tPA (+) or vehicle (control; −). (B) Representative microphotograph of Wt cerebral cortical neurons incubated during 5 minutes with vehicle (control; a, c, e, and g) or 5 nmol/L of tPA (b, d, f, and h), and stained with antibodies against the dendritic marker anti-microtubule associated protein (MAP-2) (red) and pAMPK (green). Magnification × 40 in (a, b) and × 60 in (c–h). (C) Representative microphotograph of Wt cerebral cortical neurons incubated 5 minutes with 5 nmol/L of tPA and stained with antibodies against synaptophysin (red) and pAMPK (green). Arrows denote examples where presynaptic synaptophysin-positive vesicles are in juxtaposition with postsynaptic pAMPK. Magnification × 100. (D) Representative western blot analysis of pAMPK expression in cerebral cortical synaptoneurosomes prepared from the cerebral cortex of Wt mice after transient middle cerebral artery occlusion (tMCAO) and treatment with either 0.9 mg/kg/IV of recombinant tPA (rtPA) or a comparable volume of saline solution. (E) Representative western blot analysis of the expression NR2A and NR2B subunits of NMDARs in synaptoneurosomes prepared from the cerebral cortex of Wt mice. (F) Representative western blot analysis of pAMPK expression in cerebral cortical synaptoneurosomes prepared from the cerebral cortex of Wt mice 5 minutes after tMCAO and treatment with 0.9 mg/kg/IV of rtPA alone or in combination with 2 μg of MK-801.

Article Snippet: Recombinant murine tPA, proteolytically inactive tPA (itPA) with an alanine for serine substitution at the active site Ser481 (S481A), human Lys plasmin, an ELISA kit that detects active tPA, and sheep anti-tPA antibodies (Cat # SASMTPA) were acquired from Molecular Innovations (Novi, MI, USA).

Techniques: Activation Assay, Western Blot, Expressing, Incubation, Staining, Marker, Recombinant

Journal: Circulation

Article Title: Releasing the Brakes on the Fibrinolytic System in Pulmonary Emboli: Unique Effects of Plasminogen Activation and α2-Antiplasmin Inactivation

doi: 10.1161/CIRCULATIONAHA.116.024421

Figure Lengend Snippet: Lung tissue containing FITC-fibrin labeled (green) pulmonary emboli was immunostained (red) to detect the expression of (A) tPA, (B) uPA and (C) plasminogen (Pg) (D) The total immune-stained area (arbitrary units; a.u) for each protein was measured in a 20× (100 μm) image of an embolized vs non embolized (Control; dashed yellow outline) pulmonary artery in the lungs. n=3, mean ± SEM. **p<0.01; ns, non-significant.

Article Snippet: The primary antibodies include rabbit anti-mouse against tPA (ASMTPA-GF; Molecular Innovations), uPA (urokinase plasminogen activator) (ASMUPA-GF-HT; Molecular Innovations) and PAI-1 (IASMPAI-GF; Innovative Research), rabbit anti-mouse plasminogen (Abcam), goat anti-mouse α2-antiplasmin (AF1239; R&D Systems), and rat anti-mouse Ly6G (clone1A8, #127602; Biolegend).

Techniques: Labeling, Expressing, Staining