Journal: Developmental biology

Article Title: Genetic Dissection of Midbrain Dopamine Neuron Development in vivo

doi: 10.1016/j.ydbio.2012.09.019

Figure Lengend Snippet: Quantification of cells in control and mutant mice reveals significant differences in the average number of TH+ and double positive neurons in the VTA and SNc. The average number of TH+ neurons and the 95% confidence intervals of counts from the VTA (A, B) and SNc (C, D) of control (dark grey shaded bars) and mutant mice (dark brown bars) in ventral, intermediate and dorsal planes. (A) The average number of TH+/Calb+ neurons in the VTA of mutant mice (light brown bars) was significantly lower than that of control mice (light grey bars) in all planes quantified. Calb+ neurons in the VTA indicated that the medial domain was not devoid of cells. (B) There were no double immunolabeled TH+/GIR2K+ neurons observed in the ventral VTA of any of the mutant GIRK2-stained slices consistent with the loss of VTA. (C) Although TH+ neurons were diminished in SNc, the average number of TH+/Calb+ neurons in the SNc was not significantly different between controls (light grey bars) and mutants (light brown bars) at any of the three horizontal planes. The blue arrowheads points to the average of TH+/Calb+ neurons. (D) The average number of TH+/GIRK2+ neurons in the SNc of mutant mice was significantly lower than that of control mice in all planes quantified. Images in A, C display TH+ (green), Calb+ (red) and TH+/Calb+ (yellow) cells while images in B, D display TH+ (green), GIRK2+ (red) and TH+/GIRK2+ (yellow) cells. Gray shading of bars indicates data from control animals with dark gray bars corresponding to average TH+ cell counts and nested light gray bars corresponding to average double positive cell counts. Brown shading indicates data from mutant animals with dark brown bars corresponding to average TH+ cell counts and nested lighter brown bars corresponding to average double positive cell counts.

Article Snippet: Calb indin (CALB) and g -protein i nward r ectifying potassium ( K ) channel 2 (GIRK2) were detected with anti-CALB (Swant; 1:1000) or anti-Girk2 (Alomone labs; 1:80), respectively.

Techniques: Mutagenesis, Immunolabeling, Staining

Journal: The Journal of Neuroscience

Article Title: Melanocortin-4 Receptor Regulates Hippocampal Synaptic Plasticity through a Protein Kinase A-Dependent Mechanism

doi: 10.1523/JNEUROSCI.3282-12.2013

Figure Lengend Snippet: Activation of endogenous MC4R enhances maturation of functional synapses and neurotransmission. a–c, Silencing of MC4R abolished the increase of mature spines and GluA1-containing spines by d-Tyr MTII (1 μm). Hippocampal neurons were cotransfected with GFP plasmid together with shMC4R and mKOrange constructs at 17–18 DIV as indicated. The neurons at 21–22 DIV were treated with d-Tyr MTII for 2 h. a, Representative images. Scale bars: top row, 10 μm; bottom rows, 5 μm. b, c, Quantification of mature spines (b) and SEP-GluA1-containing spines (c). Data were expressed as mean ± SEM; ***p < 0.001, d-Tyr MTII versus control (Con), two-way ANOVA. d–g, MC4R knockdown abolished the d-Tyr MTII-stimulated increase in neurotransmission. Hippocampal neurons were cotransfected with GFP construct together with or without shMC4R and then treated with d-Tyr MTII. d, Representative mEPSC traces. e–g, Cumulative distribution of interevent intervals (inversely proportional to frequency (e), and quantification of frequency (f), and amplitude (g) of mEPSCs. GFP-expressing neurons in the control, the shMC4R-transfected conditions, and their neighboring untransfected neurons (non GFP) were recorded. Data were presented as mean ± SEM; ***p < 0.001, d-Tyr MTII versus Control treatment, two-way ANOVA; ###p < 0.001 versus GFP cells in shMC4R (with d-Tyr MTII treatment), one-way ANOVA with Student-Newman–Keuls test (f); one-way ANOVA with Kruskal–Wallis test (g).

Article Snippet: The antibodies used include the following: anti-MC4R (N terminus; Alomone Labs); anti-pSer845 GluA1 and anti-GluA1 antibodies (PhosphoSolutions); anti-GluA1 (N terminus; Calbiochem); anti-pSer831 GluA1 (Millipore Bioscience Research Reagents). d -Tyrosine melanotan II ( d -Tyr MTII) was purchased from Phoenix Pharmaceuticals, MTII from Tocris Bioscience, and H89 from Sigma or Tocris Bioscience.

Techniques: Activation Assay, Functional Assay, Plasmid Preparation, Construct, Expressing, Transfection

Journal: The Journal of Neuroscience

Article Title: Melanocortin-4 Receptor Regulates Hippocampal Synaptic Plasticity through a Protein Kinase A-Dependent Mechanism

doi: 10.1523/JNEUROSCI.3282-12.2013

Figure Lengend Snippet: MC4R regulates surface levels of GluA1 through PKA-dependent phosphorylation. a–d, Hippocampal neurons were incubated with d-Tyr MTII (100 nm) and H89 (10 μm) for 2 h and then stained for surface GluA1. a, Representative images. Scale bar, 10 μm. b–d, Quantification of density (b), size (c), and intensity (d) of surface GluA1 clusters. Data were expressed as mean ± SEM, **p < 0.01, d-Tyr MTII versus control (two-way ANOVA); n = 10 neurons from each experiment, two experiments). e, Surface and total proteins of hippocampal neurons after d-Tyr MTII treatment were collected and subjected to Western blot analysis for GluA1. f, Fold change (three experiments; *p < 0.05 versus 0 min, one-way ANOVA with Student-Newman–Keuls test. g, h, Hippocampal neurons were cotreated with d-Tyr MTII and H89 for 1 h. g, Western blot analysis. h, Quantitative analysis; *p < 0.05 d-Tyr MTII (+) versus no treatment (−), two-way ANOVA; ##p < 0.01, versus d-Tyr MTII, one-way ANOVA with Student-Newman–Keuls test. i, d-Tyr MTII increased level of pSer845 GluA1 in cultured hippocampal slices (7 DIV).

Article Snippet: The antibodies used include the following: anti-MC4R (N terminus; Alomone Labs); anti-pSer845 GluA1 and anti-GluA1 antibodies (PhosphoSolutions); anti-GluA1 (N terminus; Calbiochem); anti-pSer831 GluA1 (Millipore Bioscience Research Reagents). d -Tyrosine melanotan II ( d -Tyr MTII) was purchased from Phoenix Pharmaceuticals, MTII from Tocris Bioscience, and H89 from Sigma or Tocris Bioscience.

Techniques: Incubation, Staining, Western Blot, Cell Culture

Journal: Journal of Cellular and Molecular Medicine

Article Title: TRPC1 is regulated by caveolin‐1 and is involved in oxidized LDL‐induced apoptosis of vascular smooth muscle cells

doi: 10.1111/j.1582-4934.2008.00593.x

Figure Lengend Snippet: TRPC1 expression is linked to caveolin‐1. TRPC1 expression is correlated with caveolin‐1 expression. (A), TRPC1 and caveolin‐1 expression were analysed in human primary VSMC (hSMC, passage 5), in SMC stably expressing caveolin‐1 cell line (SMC/cav1) and in control SMC expressing an empty vector (SMC/ev), by immunoblotting using anti‐TRPC1 and anti‐caveolin‐1 antibodies as described under ‘Materials and methods’. The graph represents values of caveolin‐1 and TRPC1 band intensity after normalization for β‐actin by densitometry (a.u., arbitrary units). Results are representative of at least three independent experiments. (*P < 0.05, **P < 0.01). (B), TRPC1 expression under caveolin‐1 silencing. Expression of TRPC1 protein was analysed by immunoblotting using an anti‐TRPC1 antibody as described under ‘Materials and methods’, after transfection of SMC/cav1 with 100 nM caveolin‐1 siRNA (siRNA cav1) for 48 and 72 hrs or 100 nM scrambled siRNA (siRNA sc). The graph represents values of caveolin‐1 and TRPC1 band intensity after normalization for β‐actin by densitometry (a.u., arbitrary units). Results are representative of at least three independent experiments. (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: The following antibodies were used: polyclonal anti‐TRPC1 (Alomone Labs, Jerusalem, Israel), polyclonal anti‐caveolin‐1 and monoclonal anti‐β‐actin (Upstate Biotechnology, Billerica, MA, USA).

Techniques: Expressing, Stable Transfection, Plasmid Preparation, Western Blot, Transfection

Journal: Journal of Cellular and Molecular Medicine

Article Title: TRPC1 is regulated by caveolin‐1 and is involved in oxidized LDL‐induced apoptosis of vascular smooth muscle cells

doi: 10.1111/j.1582-4934.2008.00593.x

Figure Lengend Snippet: Caveolin‐1 silencing reduced oxLDL‐induced cell death. Caveolin‐1 silencing shifted oxLDL‐induced cell apoptosis. SMC/cav1 (A) or human primary VSMC (passage 5) (B) were either transfected with 100 nM caveolin‐1 siRNA (siRNA cav1) or with 100 nM scrambled siRNA (siRNA sc) then serum starved, and incubated with oxLDL (100 μg ApoB/ml) for 18 hrs. Expression of caveolin‐1 protein was analysed by immunoblotting using an anti‐caveolin‐1 antibody as described under ‘Materials and methods’ and cell viability was evaluated by the MTT assay. Results are expressed as percentage of untreated control and represent the mean ± S.E.M. of three independent experiments (*P < 0.05).

Article Snippet: The following antibodies were used: polyclonal anti‐TRPC1 (Alomone Labs, Jerusalem, Israel), polyclonal anti‐caveolin‐1 and monoclonal anti‐β‐actin (Upstate Biotechnology, Billerica, MA, USA).

Techniques: Transfection, Incubation, Expressing, Western Blot, MTT Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: TRPC1 is regulated by caveolin‐1 and is involved in oxidized LDL‐induced apoptosis of vascular smooth muscle cells

doi: 10.1111/j.1582-4934.2008.00593.x

Figure Lengend Snippet: OxLDL induce translocation of TRPC1 into caveolar compartment. (A), TRPC1 expression after oxLDL treatment. SMC/cav1 were incubated with oxLDL (100 μg ApoB/ml) for 10 hrs and expression of TRPC1 protein was analysed by immunoblotting using an anti‐TRPC1 antibody as described under ‘Materials and methods’. Results are representative of at least three independent experiments. (B), Increase in surface‐expressed TRPC1 induced by oxLDL. SMC/cav1 were incubated with oxLDL (100 μg ApoB/ml) for 8 or 14 hrs, then treated with sulfo‐NHS‐SS‐Biotin (1 mg/ml) and lysed. Biotinylated proteins were recovered as described under ‘Materials and methods’, and TRPC1 expression analysed by immunoblotting using an anti‐TRPC1 antibody. Total lysates were analysed with TRPC1 antibody to insure for equal loading of proteins. Results are representative of at least three independent experiments. (C), Translocation of TRPC1 into caveolae membranes upon oxLDL stimulation. SMC/cav1 were treated with oxLDL (100 μg ApoB/ml), after 8 hrs, cells were lysed in carbonate buffer and subjected to sucrose gradient sedimentation, as described under ‘Materials and methods’. A total of 50 μl of pooled fractions (4, 5 and 6 = L, light‐density fractions); 7, 8 and 9 = H1, high‐density fractions; 10, 11 and 12 = H2, high‐density fractions) were used for the detection of TRPC1 and caveolin‐1 by immunoblotting using anti‐TRPC1 and anti‐caveolin‐1 antibodies. The graph represents values of caveolin‐1 and TRPC1 band intensity by densitometry (a.u., arbitrary units). Results are representative of three independent experiments. (*P < 0.05, significantly different mean expression value from untreated conditions).

Article Snippet: The following antibodies were used: polyclonal anti‐TRPC1 (Alomone Labs, Jerusalem, Israel), polyclonal anti‐caveolin‐1 and monoclonal anti‐β‐actin (Upstate Biotechnology, Billerica, MA, USA).

Techniques: Translocation Assay, Expressing, Incubation, Western Blot, Sedimentation