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Figure Lengend Snippet: Assessment of several terminal protein expression levels. Relative protein expression levels of (A) VMAT2 (n = 7 in both groups), (B) Synaptigyrin-3 (aGH: n = 6; aSI: n = 5), (C) Syntaxin-1 (aGH: n = 7; aSI: n = 6), and (D) Munc13-3 (aGH: n = 8; aSI: n = 8) were measured. None of these proteins had different expression levels in aGH and aSI rats. (insets) The representative Western blot images, with the respective protein and actin for comparison. Group housed, aGH, blue; Socially isolated, aSI, red.

Article Snippet: Subsequently, blots were incubated with agitation for 2 h at room temperature in TBS-T/5% bovine serum albumin (05470; Sigma-Aldrich) solution containing the following primary antibody concentrations: VAMT2 (1:2000; AB1598P; Millipore Sigma); Synaptogyrin-3 (1:1000; ab106460; abcam); Syntaxin-1 (1:1000; ANR-002; Alomone laboratories).

Techniques: Expressing, Western Blot, Isolation

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: Analysis, Staining, Co-Immunoprecipitation Assay, Immunofluorescence

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: Inactivation, Expressing, Activation Assay, Produced, Patch Clamp

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, Confocal, Translocation Assay, Clinical Proteomics, Membrane, Staining, Labeling, Co-Immunoprecipitation Assay, Transfection, Western Blot, Immunofluorescence, Patch Clamp