Ryr1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Direct association of the reticulon protein RTN1A with the ryanodine receptor 2 in neurons"
Article Title: Direct association of the reticulon protein RTN1A with the ryanodine receptor 2 in neurons
Journal: Biochimica et Biophysica Acta
Figure Legend Snippet: GST-RTN1 523 inhibits specific [ 3 H]ryanodine binding to rat brain synaptosomes. (A) Equilibrium [ 3 H]ryanodine binding to rat forebrain synaptosomal membrane preparations was carried out in binding buffer containing 10 nM [ 3 H]ryanodine at the indicated free calcium concentrations in control conditions ( closed circles ) and in the presence of 0.1 μM GST ( closed triangles ) or 0.1 μM GST-RTN1 523 ( open squares ). [Ca 2 + ] was maintained, in the range 0.01 μM–1 mM, by a combination of EGTA and CaCl 2 . Free Ca 2 + concentrations were calculated as described in material and methods. Data points shown are the mean ± S.E.M., from three separate experiments performed in triplicates. (B) [ 3 H]ryanodine binding in the presence of 0.1 μM GST or GST-RTN1 523 is presented as percent of control. No specific [ 3 H]ryanodine binding was observed at 0.01 μM Ca 2 + in the presence of GST or GST-RTN1 523 . Difference in [ 3 H]ryanodine binding was plotted as percent decrease in specific binding. Data points shown are the mean ± S.E.M., from three separate experiments (* p = 0.011 by Student's t -test). (C) RyR2 evoked Ca 2 + oscillations in HEK293. Upper and lower left panels represent Fura-2 ratio time-courses of single cells expressing RyR2 together with mcherry, mcherry-RTN1A or EGFP-RTN4A. Cells were continuously perfused with buffer containing 0 mM Ca 2 + (nominal free), 1 mM Ca 2 + and 0 mM Ca 2 + + 10 mM caffeine as indicated by the bars at the top. Lower right panel shows a quantitative analysis performed by integration of the respective single peak areas referred to area under curve for estimation of the total amount of the cytosolic [Ca 2 + ] arising through RyR2 dependent Ca 2 + oscillations. The fraction of cells that showed both oscillations as well as a clear caffeine peak in comparison to those that lacked oscillations before a single caffeine peak are given in percentages in the graph. A two-sample t-test was carried out to test for significance as indicated by the p values at the bottom of the panel.
Techniques Used: Binding Assay, Expressing
Figure Legend Snippet: Immunohistochemical distribution pattern of RTN1A and RyR2 in rat hippocampus and cerebellum. Representative staining patterns for RTN1A (A,C,D) and RyR2 (B,E) on sections of rat hippocampus (A,B) and cerebellar cortex (C,D,E). In the hippocampus, immunoreactivity for both proteins is found in granule cells (G), mossy fiber axons, and in stratum lucidum (SL). In the cerebellum, RTN1A-immunoreactivity was confined to Purkinje cell bodies (P), their dendrites in ML (C; arrow) and axons (D; arrowheads). (E) Confocal immunofluorescent image showing RyR2 staining in Purkinje cells. Unlike RTN1A, RyR2 was also found in granule cell layer (Gl). Scale bars: A and B, 500 μm; H, hilus; G, Granule cell layer; So, stratum oriens; Sr, stratum radiatum; Slm, stratum lacunosum molecular; Iml, inner molecular layer; M + Oml, Middle outer molecular layer; CA1-3, Cornu ammonis; SL, stratum lucidum; S, Subiculum; Pr, Presubiculum; Pa, Parasubiculum.
Techniques Used: Immunohistochemistry, Staining
Figure Legend Snippet: Identification of RyR2 as a binding partner of RTN1A. (A) Schematic representation of full length RTN1A and the GST-RTN1 523 construct ( right ). RHD: reticulon homology domain; TM1 and TM2: transmembrane domain 1 and 2. GST pull-downs were performed with GST-RTN1 523 using detergent-solubilized mouse brain proteins. GST or empty glutathione beads served as negative controls, whereas GST-NiR was tested to control for GST-RTN1 523 binding specificity. Arrow denotes protein band that was consistently pulled down with GST-RTN1 523 and from which RyR2 was identified by mass spectrometry. Note that this band is absent in the different control samples. Asterisks indicate the GST fusion proteins and their relative amounts used in the GST pull-down. The silver stained gel shown is representative of three independent experiments. (B) Upper panel , Western Blot of samples from (A) probed with RyR2 antibody. Note the double-band that is identified as RyR2. The higher molecular mass band corresponds to intact RyR2, while the lower band presumably represents a proteolytic degradation fragment of RyR2. The intensity of the RyR2 bands increase with increasing amounts of GST-RTN1 523 . Lower panel , shows Ponceau S staining of the pull-downs to assess the relative amounts of each GST fusion protein. Input lane shows one-twentieth of the amount used for pull-down. WB, Western blot. The Western blot shown is representative of three independent experiments.
Techniques Used: Binding Assay, Construct, Mass Spectrometry, Staining, Western Blot
Figure Legend Snippet: Immunocytochemical distribution of mCherry-RTN1 523 and RyRs in HEK293 cells. (A) HEK293 cells were transiently transfected with mCherry-RTN1 523 in the absence (a) or presence of untagged RyR2 (d–f) or RyR1 (g–i). Single-transfections of untagged RyR2 (b) and RyR1 (c) were carried out as a comparison. Cells were immunostained with anti-RyR2 (b,d–f) or anti-RyR1 (c,g-i) antibodies and visualized by confocal microscopy. Note that single-transfected mCherry-RTN1 523 is uniformly distributed in the cytosol (a), but altered to a more reticular staining pattern when co-expressed with RyR2 (d–f). mCherry-RTN1 523 remained uniformly distributed in cells cotransfected with RyR1 (g–i). Cells shown represent at least 20 representative cells per condition. (B) Immunofluorescence confocal microscopy analysis of RyR ER localization in HEK293 cells. HEK293 cells were single-transfected with untagged RyR1 cDNA or RyR2 cDNA, immunostained with the indicated antibodies and imaged using immunofluorescence microscopy to demonstrate RyR and calreticulin (ER-specific marker) colocalization. Cells were immunostained for RyR isoforms (green) and Calreticulin (red), respectively. Space bar: 10 μm. (C) Extent of colocalization between mCherry-RTN1 523 and RyR isoforms was quantified using Pearson correlation coefficient and determined through correlation analysis with Leica SP5 software from 10 different cells per group. (*** p = 0.003 by Student's t -test).
Techniques Used: Transfection, Confocal Microscopy, Staining, Immunofluorescence, Microscopy, Marker, Software
Figure Legend Snippet: RTN1A associates preferentially with RyR2 channel in vivo . (A) Left panels , detergent-solubilized protein from HEK293 cells transiently transfected with untagged RyR2 plus RTN1A-myc was immunoprecipitated (IP) with rabbit polyclonal anti-RTN1A antibodies or control rabbit IgG. Immunoprecipitated proteins were detected on immunoblots with monoclonal anti-RyR2 or anti-RTN1A antibodies. Note that native HEK293 cells do not express endogenous levels of RTN1A or RyR2. Right panels , detergent-solubilized protein from HEK293 cells transiently transfected with untagged RyR2 plus RTN4A-myc was immunoprecipitated with rabbit polyclonal anti-RTN4 antibodies or control rabbit IgG. Immunoprecipitated proteins were detected on immunoblots with monoclonal anti-RyR2 or anti-RTN4A antibodies. Input lane shows one-eighth of the amount used for immunoprecipitation. WB, Western blot. (B) Detergent-solubilized protein from rat cerebellum was used for co-immunoprecipitations with rabbit anti-RTN1A, rabbit anti-RTN1A, or control rabbit IgG. Immunoprecipitated proteins were resolved by SDS-PAGE blotted on PVDF membranes and probed with monoclonal antibodies as indicated at the right. Input lane shows one-tenth of the amount used for immunoprecipitation. WB, Western blot. (C) Detergent-solubilized protein from rat cerebellum was used for co-immunoprecipitations with mouse anti-RyR2, mouse anti-RTN1A, mouse anti-RTN1A, or control mouse IgG. Immunoprecipitated proteins were resolved by SDS-PAGE, blotted on PVDF membranes and probed with antibodies as indicated at the right. Input lane shows one-tenth of the amount used for immunoprecipitation. WB, Western blot. (D) Detergent-solubilized protein from rat cerebellum was immunoprecipitated with mouse anti-RTN1A, mouse anti-RyR2, or control mouse IgG. Immunoprecipitated proteins were resolved by SDS-PAGE, blotted on PVDF membranes and probed with antibodies as indicated at the right. Note that RyR1 co-immunoprecipitates with mouse anti-RyR2, but not with mouse anti-RTN1A antibodies. Input lane shows one-fifth of the amount used for immunoprecipitation. WB, Western blot.
Techniques Used: In Vivo, Transfection, Immunoprecipitation, Western Blot, SDS Page
Figure Legend Snippet: Identification of the RyR2 binding domain. (A) Schematic representation of rat RTN1A protein and RTN1A fragments used to construct GST fusion proteins for the pull-down experiments. HHD: high homology domain; LNT: long N-terminal fragment. (B) GST-RTN1 fragments were used as baits in pull-down experiments using detergent-solubilized mouse brain proteins. GST, GST-NiR or empty glutathione beads served as negative controls. Binding of RyR2 was subsequently detected by immunoblot ( upper panel ). Ponceau S staining of the pull-downs shows the relative amounts of each GST fusion protein ( lower panel ). Input lane shows one-tenth of the amount used for immunoprecipitation. WB, Western blot. Results are representative of three independent experiments.
Techniques Used: Binding Assay, Construct, Staining, Immunoprecipitation, Western Blot