Journal: Journal of Biological Chemistry

Article Title: A Cav3.2/Syntaxin-1A Signaling Complex Controls T-type Channel Activity and Low-threshold Exocytosis

doi: 10.1074/jbc.m111.290882

Figure Lengend Snippet: FIGURE1.Cav3.2channelinteractswithsyntaxin-1Aincentralneurons.A,confocalimagesofnRTneuronspermeabilizedandstainedforCav3.2(green)and syntaxin-1A (red). Overlaid images and colocalized pixels (in white) are shown. B, intensity correlation analysis (ICA) plots of Cav3.2 and syntaxin-1A staining intensities against their respective (A-a)(B-b) values performed from the ROIs indicated in the overlaid image in A. IQC 0.31 and 0.38 for soma and neurite regions, respectively. C, co-immunoprecipitation of syntaxin-1A from rat brain homogenate with specific anti-Cav3.1, anti-Cav3.2, and anti-Cav3.3 antibodies.

Article Snippet: Affinity-purified, rabbit polyclonal antibodies recognizing Cav3.1, Cav3.2, and Cav3.3 were purchased from Alomone Lab andmousemonoclonal syntaxin 1A antibody was fromChemicon International (Temecula, CA).

Techniques: Staining, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: A Cav3.2/Syntaxin-1A Signaling Complex Controls T-type Channel Activity and Low-threshold Exocytosis

doi: 10.1074/jbc.m111.290882

Figure Lengend Snippet: FIGURE 2. Syntaxin-1A modulates Cav3.2 channel inactivation. A, repre- sentative Ba2 current traces recorded from a Cav3.2- (top panel) and Cav3.2/ Stx-1A-expressing cell (bottom panel) in response to 150 ms depolarizing steps to 20 mV from a holding potential varied from 120 mV to 50 mV (a to f). B, corresponding mean normalized steady-state inactivation curves for Cav3.2- (filled circles) and Cav3.2/Stx-1A-expressing cells (open circles). C, plot of the mean shift values in the half-inactivation potential of Cav3.2 channel coexpressed with the different protein combinations indicated in the figure. D, mean normalized activation curve for Cav3.2 (filled circles) and Cav3.2/ Stx1A-expressing cells (open circles). Inset indicates the shift values in the half- activation potential produced upon coexpression of Stx1A or Stx1ATM. Stx- 1A, syntaxin-1A; BoNT/C, botulinium neurotoxin C1.

Article Snippet: Affinity-purified, rabbit polyclonal antibodies recognizing Cav3.1, Cav3.2, and Cav3.3 were purchased from Alomone Lab andmousemonoclonal syntaxin 1A antibody was fromChemicon International (Temecula, CA).

Techniques: Expressing, Activation Assay, Produced

Journal: Journal of Biological Chemistry

Article Title: A Cav3.2/Syntaxin-1A Signaling Complex Controls T-type Channel Activity and Low-threshold Exocytosis

doi: 10.1074/jbc.m111.290882

Figure Lengend Snippet: FIGURE 3. Syntaxin-1A interacts within the carboxy-terminal domain of Cav3. 2. A, schematic representation of the different constructs of intracellular regions of Cav3.2 used. B, whole-cell Ba2 currents (top panels) recorded in response to a 150 ms depolarizing step to 20 mV from a holding potential of 85 mV before (P1) and after (P2) a 5 s hyperpolarizing pulse to 130 mV in a Cav3.2 (left panel), Cav3.2/Stx-1A (middle panel), and Cav3.2/Stx-1A/CD4-Cav3.2Cter- expressing cell (right panel) and the corresponding mean plot of the current facilitation (IP2/IP1) (bottom panel). Note that the hyperpolarizing pulse produces a strong current facilitation in the presence of Stx-1A, which is competitively and specifically abolished upon co-expression of the CD4-Cav3.2Cter construct. C, confocal images of living COS cells showing the translocation of the EGFP-Cav3.2Cter construct (green) to the plasma membrane mediated by Stx-1A. Plasma membrane was stained with rhodamine-labeled concanavalin A (ConA-Rhod, red). Overlaid images and pixel intensity profiles of crossed sections indicated by the white line are shown. Note that Stx-1A does not translocate EGFP-Cav3.2II-III linker fusion protein. D, co-immunoprecipitation of the CD4-EGFP-Cav3.2Cter fusion protein from tsA-201 cells co-transfected with Stx-1A-Myc. The upper panel shows the immunoblot of CD4-EGFP-Cav3.2Cter fusion protein in the absence () and presence () of Stx-1A-Myc using an anti-GFP antibody. *, possible degradation of the CD4-EGFP-Cav3.2Cter fusion protein. The lower panel shows the results of the co-immunoprecipitation of the CD4-EGFP-Cav3.2Cter fusion protein with Stx-1A-Myc using an anti-Myc antibody. In the absence of Stx-1A-Myc, the antibody alone is not able to immunoprecipitate the CD4-EGFP-Cav3.2Cter fusion protein.

Article Snippet: Affinity-purified, rabbit polyclonal antibodies recognizing Cav3.1, Cav3.2, and Cav3.3 were purchased from Alomone Lab andmousemonoclonal syntaxin 1A antibody was fromChemicon International (Temecula, CA).

Techniques: Construct, Expressing, Translocation Assay, Clinical Proteomics, Membrane, Staining, Labeling, Immunoprecipitation, Transfection, Western Blot

Journal: PLoS ONE

Article Title: Rivastigmine Lowers Aβ and Increases sAPPα Levels, Which Parallel Elevated Synaptic Markers and Metabolic Activity in Degenerating Primary Rat Neurons

doi: 10.1371/journal.pone.0021954

Figure Lengend Snippet: Levels of low molecular weight neuronal sAPP (LMW-sAPP), high molecular weight glial sAPP (HMW-sAPP), and sAPPα were compared to levels of the presynaptic protein markers SNAP-25 and syntaxin-4, and the postsynaptic protein marker PSD-95. All values are expressed as a % of vehicle-treated cells for comparison. PSD-95 and SNAP-25 levels increased dose-dependently with rivastigmine treatment (both p<0.05), and syntaxin-4 levels increased but this change did not reach significance (7A and 7B). LMW-sAPP was increased with 5 µM rivastigmine but plateaued at the higher concentration. HMW-sAPP increased to a lesser extent at 5 µM, but was similar to vehicle at the higher concentration (7C). These data suggest that increased neuronal and decreased glial sAPP may be involved in the enhanced neuronal and synaptic marker stability that results from rivastigmine treatment, and that modulation of α-secretase may be involved in these effects.

Article Snippet: Blots of the lysates were probed with mouse-anti-SNAP-25 (Millipore), mouse-anti-PSD-95 (Antibodies Incorporated, Davis, CA), mouse-anti-syntaxin-4 (BD Transduction Labs, Franklin Lakes, NJ), mouse anti-β-actin (Sigma-Aldrich), and the 22C11 antibody as above, followed by the appropriate secondary antibody and ECL detection.

Techniques: Molecular Weight, Marker, Concentration Assay