Journal: Journal of Pineal Research

Article Title: Overexpression of melatonin membrane receptors increases calcium‐binding proteins and protects VSC4.1 motoneurons from glutamate toxicity through multiple mechanisms

doi: 10.1111/j.1600-079x.2012.01022.x

Figure Lengend Snippet: Fig. 1. Melatonin receptor (MT1 and MT2) overexpression prevented ventral spinal cord 4.1 (VSC4.1) motoneuron death. Plasmid-mediated increase (P›) in expression was shown. Treatment groups: control cells (Con), 100 nm melatonin (24 h pretreatment), MT1-overexpressed cells, MT2-overexpressed cells, MT1 + MT2–overexpressed cells, G-protein receptor 50 (GPR50)-overexpressed cells, 25 lm L-glutamate (LGA) (24 h exposure), 100 nm melatonin (24 h pretreatment) + 25 lm LGA (24 h), MT1-overexpressed cells + 25 lm LGA (24 h), MT2-over- expressed cells + 25 lm LGA (24 h), MT1 + MT2–overexpressed cells + 25 lm LGA (24 h), GPR50-overexpressed cells + 25 lm LGA (24 h).(A) ApopTag assayshowingrepresentative imagesfromeach treatmentgroup.Arrowsindicateapoptoticcells. (B)Bargraphsindicating the percentage of apoptotic cells (ApopTag assay) and viability (trypan blue dye exclusion test) counted from each group. (C) Representative pictures show levels of MT1, MT2, GPR50, and b-actin levels (Western blotting). (D) Bar graphs indicating the changes in expression of MT1, MT2, and GPR50 over Con [Correction added on 8 Oct 2012, after first online publication: Fig. 1 Panel A has been corrected].

Article Snippet: After washing with serum-free standard medium (DMEM/F12), motoneurons were transfected in 1% low serum medium [26] over the course of 24 h at 37 C with 35 lL lipofectamine (Life Technologies, Grand Island, NY, USA) and 10 lg of GFP-tagged ORF clone of homosapiens MT1 (MTNR1A: RG210385), MT2 (MTNR1B: RG216445), or GPR50 (RG217839) plasmid (Origene, Rockville, MD, USA).

Techniques: Over Expression, Plasmid Preparation, Expressing, Control, Western Blot

Journal: Journal of Pineal Research

Article Title: Overexpression of melatonin membrane receptors increases calcium‐binding proteins and protects VSC4.1 motoneurons from glutamate toxicity through multiple mechanisms

doi: 10.1111/j.1600-079x.2012.01022.x

Figure Lengend Snippet: Fig. 2. Overexpression of MT1 and MT2 increases calbindin D28K and parvalbumin for suppressing Ca2+ rise and calpain:calpastatin ratio in ventral spinal cord 4.1 (VSC4.1) cells. Plasmid-mediated increase (P›) in expression was shown. Treatment groups: control cells (Con), 100 nm melatonin (24 h pretreatment), MT1-overexpressed cells, MT2-overexpressed cells, MT1 + MT2–overexpressed cells, G- protein receptor 50 (GPR50)-overexpressed cells, 25 lm L-glutamate (LGA) (24 h), 100 nm melatonin (24 h pretreatment) + 25 lm LGA (24 h exposure), MT1-overexpressed cells + 25 lm LGA (24 h exposure), MT2-overexpressed cells + 25 lm LGA (24 h exposure), MT1 + MT2–overexpressed cells + 25 lm LGA (24 h exposure), GPR50-overexpressed cells + 25 lm LGA (24 h exposure). (A) Determination of intracellular free Ca2+ levels at 24 h. (B) Western blot analysis to show levels of calbindin D28K, parvalbumin, calpain, calpastatin, and b-actin. (C) Bar graphs indicating the changes in expression of calbindin D28K and parvalbumin over Con. (D) Densi- tometric analysis showing the calpain:calpastatin ratio.

Article Snippet: After washing with serum-free standard medium (DMEM/F12), motoneurons were transfected in 1% low serum medium [26] over the course of 24 h at 37 C with 35 lL lipofectamine (Life Technologies, Grand Island, NY, USA) and 10 lg of GFP-tagged ORF clone of homosapiens MT1 (MTNR1A: RG210385), MT2 (MTNR1B: RG216445), or GPR50 (RG217839) plasmid (Origene, Rockville, MD, USA).

Techniques: Over Expression, Plasmid Preparation, Expressing, Control, Western Blot

Journal: Journal of Pineal Research

Article Title: Overexpression of melatonin membrane receptors increases calcium‐binding proteins and protects VSC4.1 motoneurons from glutamate toxicity through multiple mechanisms

doi: 10.1111/j.1600-079x.2012.01022.x

Figure Lengend Snippet: Fig. 3. Overexpression of MT1 and MT2 increase estrogen recep- tor (ERb:ERa) ratio and suppression inflammatory factors in ventral spinal cord 4.1 (VSC4.1) cells. Plasmid-mediated increase (P›) in expression was shown. Treatment groups: control cells (Con), 100 nm melatonin (24 h pretreatment), MT1-overexpressed cells, MT2-overexpressed cells, MT1 + MT2–overexpressed cells, G-protein receptor 50 (GPR50)-overexpressed cells, 25 lm L-glu- tamate (LGA) (24 h), 100 nm melatonin (24 h pretreat- ment) + 25 lm LGA (24 h), MT1-overexpressed cells + 25 lm LGA (24 h), MT2-overexpressed cells + 25 lm LGA (24 h), MT1 + MT2–overexpressed cells + 25 lm LGA (24 h), GPR50- overexpressed cells + 25 lm LGA (24 h). (A) Western blot anal- ysis to show levels of ERb, ERa, NF-jB, COX-2, and b-actin. (B) Densitometric analysis showing the ERb: ERa ratio. (C) Bar graphs indicating the changes in the expression of NF-jB and COX-2 over Con.

Article Snippet: After washing with serum-free standard medium (DMEM/F12), motoneurons were transfected in 1% low serum medium [26] over the course of 24 h at 37 C with 35 lL lipofectamine (Life Technologies, Grand Island, NY, USA) and 10 lg of GFP-tagged ORF clone of homosapiens MT1 (MTNR1A: RG210385), MT2 (MTNR1B: RG216445), or GPR50 (RG217839) plasmid (Origene, Rockville, MD, USA).

Techniques: Over Expression, Plasmid Preparation, Expressing, Control, Western Blot

Journal: Journal of Pineal Research

Article Title: Overexpression of melatonin membrane receptors increases calcium‐binding proteins and protects VSC4.1 motoneurons from glutamate toxicity through multiple mechanisms

doi: 10.1111/j.1600-079x.2012.01022.x

Figure Lengend Snippet: Fig. 4 Overexpression of MT1 and MT2 increases survival and angiogenesic factors in ventral spinal cord 4.1 (VSC4.1) cells. Plas- mid-mediated increase (P›) in expression was shown. Treatment groups: control cells (Con), 100 nm melatonin (24 h pretreatment), MT1-overexpressed cells, MT2-overexpressed cells, MT1 + MT2– overexpressed cells, G-protein receptor 50 (GPR50)-overexpressed cells, 25 lm L-glutamate (LGA) (24 h exposure), 100 nm melatonin (24 h pretreatment) + 25 lm LGA (24 h exposure), MT1-overex- pressed cells + 25 lm LGA (24 h), MT2-overexpressed cells + 25 lm LGA (24 h exposure), MT1 + MT2–overexpressed cells + 25 lm LGA (24 h exposure), GPR50-overexpressed cells + 25 lm LGA (24 h exposure). (A) Western blot analysis to show levels of p-Akt, Bcl-2, p-Bad, Flk-1, Flt-1, VEGF, and b-actin. (B) Bar graphs indicating the changes in expression of p-Akt, Bcl-2, and p-Bad over Con. (C) Bar graphs indicating changes in the expression of Flk-1, Flt-1, and VEGF over Con.

Article Snippet: After washing with serum-free standard medium (DMEM/F12), motoneurons were transfected in 1% low serum medium [26] over the course of 24 h at 37 C with 35 lL lipofectamine (Life Technologies, Grand Island, NY, USA) and 10 lg of GFP-tagged ORF clone of homosapiens MT1 (MTNR1A: RG210385), MT2 (MTNR1B: RG216445), or GPR50 (RG217839) plasmid (Origene, Rockville, MD, USA).

Techniques: Over Expression, Expressing, Control, Western Blot

Journal: Journal of Pineal Research

Article Title: Overexpression of melatonin membrane receptors increases calcium‐binding proteins and protects VSC4.1 motoneurons from glutamate toxicity through multiple mechanisms

doi: 10.1111/j.1600-079x.2012.01022.x

Figure Lengend Snippet: Fig. 5. Overexpression of MT1 and MT2 suppresses apoptotic pathways in ventral spinal cord 4.1 (VSC4.1) cells. Plasmid-mediated increase (P›) in expression was shown. Treatment groups: control cells (Con), 100 nm melatonin (24 h pretreatment), MT1-overexpressed cells, MT2-overexpressed cells, MT1 + MT2–overexpressed cells, G-protein receptor 50 (GPR50)-overexpressed cells, 25 lm L-glutamate (LGA) (24 h), 100 nm melatonin (24 h pretreatment) + 25 lm LGA (24 h), MT1-overexpressed cells + 25 lm LGA (24 h), MT2-over- expressed cells + 25 lm LGA (24 h), MT1 + MT2–overexpressed cells + 25 lm LGA (24 h), GPR50-overexpressed cells + 25 lm LGA (24 h). (A) Western blot analysis to show levels of Bax, Bcl-2, active caspase-9, active caspase-3, and b-actin. (B) Densitometric analysis showing the Bax:Bcl-2 ratio. (C) Bar graphs indicating the changes in expression of active caspase-9 and active caspase-3 over Con. (D) Colorimetric determination of caspase-9 and caspase-3 activities.

Article Snippet: After washing with serum-free standard medium (DMEM/F12), motoneurons were transfected in 1% low serum medium [26] over the course of 24 h at 37 C with 35 lL lipofectamine (Life Technologies, Grand Island, NY, USA) and 10 lg of GFP-tagged ORF clone of homosapiens MT1 (MTNR1A: RG210385), MT2 (MTNR1B: RG216445), or GPR50 (RG217839) plasmid (Origene, Rockville, MD, USA).

Techniques: Over Expression, Plasmid Preparation, Expressing, Control, Western Blot

Journal: Journal of Pineal Research

Article Title: Overexpression of melatonin membrane receptors increases calcium‐binding proteins and protects VSC4.1 motoneurons from glutamate toxicity through multiple mechanisms

doi: 10.1111/j.1600-079x.2012.01022.x

Figure Lengend Snippet: Fig. 6. Silencing MT1 and MT2 enhanced glutamate toxicity in ventral spinal cord 4.1 (VSC4.1) cells. We indicated the use of RNA interference (RNAi) to cause a decrease in expression. Treatment groups: control cells (Con), 25 lm L-glutamate (LGA) (24 h exposure), MT1-silenced cells + 25 lm LGA (24 h exposure); MT2-silenced cells + 25 lm LGA (24 h), MT1 + MT2-silenced cells + 25 lm LGA (24 h exposure); G-protein receptor 50 (GPR50) silenced cells + 25 lm LGA (24 h), 25 lm LGA (24 h), 25 lm LGA + 100 nm melatonin (24 h exposure), MT1-silenced cells + 25 lm LGA+ 100 nm melatonin (24h exposure), MT2 silenced cells + 25 lm LGA + 100 nm melatonin (24 h exposure), MT1 + MT2-silenced cells + 25 lm LGA + 100 nm melatonin (24 h exposure), GPR50 silenced cells + 25 lm LGA + 100 nm melatonin (24 h exposure). (A) Representative pictures showing levels of MT1, MT2, GPR50, and b-actin levels (Western blotting). (B) Bar graphs indicating the percentage of viability (trypan blue dye exclusion test) counted from each group. (C) Colorimetric determination of caspase-3 activity.

Article Snippet: After washing with serum-free standard medium (DMEM/F12), motoneurons were transfected in 1% low serum medium [26] over the course of 24 h at 37 C with 35 lL lipofectamine (Life Technologies, Grand Island, NY, USA) and 10 lg of GFP-tagged ORF clone of homosapiens MT1 (MTNR1A: RG210385), MT2 (MTNR1B: RG216445), or GPR50 (RG217839) plasmid (Origene, Rockville, MD, USA).

Techniques: Expressing, Control, Western Blot, Activity Assay

Journal: Journal of Pineal Research

Article Title: Melatonin pretreatment alleviates the long‐term synaptic toxicity and dysmyelination induced by neonatal Sevoflurane exposure via MT1 receptor‐mediated Wnt signaling modulation

doi: 10.1111/jpi.12771

Figure Lengend Snippet: Acute effects of repeated neonatal Sev exposure on melatonin signaling in hippocampus. (A) Experimental design. (B, C) Western blotting and quantification of MT1 and MT2 receptors in the hippocampus of control and Sev‐treated mice. MT1, but not MT2 receptor was up‐regulated in Sev‐treated mice at 3 dps. (D) Levels of cAMP in hippocampus of control and Sev‐treated mice at 3 dps. (E, F) Western blotting and quantification of p‐PKA, p‐CREB, and CREB in the hippocampus of control and Sev‐treated mice at 3dps. (G, H) Double immunostaining of MT1/NeuN and MT1/NG2 in the hippocampus of control and Sev‐treated mice. Notice the expression of MT1 by neurons and oligodendrocyte precursors. N = 6 mice per group. Con, control. Sev, Sevoflurane. * p < .05. ** p < .01. *** p < .001. One‐way ANOVA (C). Student’ t test (D, F–H). Bars = 50 μm

Article Snippet: MT1 receptor knockout mice were bought from Cyagen Biosciences (Stock No. KOCMP‐17773‐Mtnr1a‐B6N).

Techniques: Western Blot, Control, Double Immunostaining, Expressing

Journal: Journal of Pineal Research

Article Title: Melatonin pretreatment alleviates the long‐term synaptic toxicity and dysmyelination induced by neonatal Sevoflurane exposure via MT1 receptor‐mediated Wnt signaling modulation

doi: 10.1111/jpi.12771

Figure Lengend Snippet: Blockage of the protective effects of melatonin on Sev‐induced synaptic impairment and dysmyelination by MT1‐shRNA (shMT1). (A) Illustration of injection of lentivirus expressing shMT1. (B, C) Western blotting and quantification of MT1 in hippocampus injected with different shMT1. ShMT1‐#2 was most effective in silencing the expression of MT1 (D) Western blotting and quantification of GluR1, GluR2, Homer, and PSD95 in mice of the following groups: control + vehicle, Sev + vehicle, Sev + mel, Sev + mel + EV (EV, empty vector), Sev + mel + shMT1. Notice that shMT1 blocked the up‐regulation of GluR1, GluR2, Homer, and PSD95 by melatonin. (E) Immunostaining of vglut1. Notice the weak immunoreactivity of vglut1 in mice treated with shMT1 as compared with that in mice treated with EV. (F) Western blotting and quantification of PDGFRα, NG2, Sox10, and Olig2 in mice of the following groups: control + vehicle, Sev + vehicle, Sev + mel, Sev + mel + EV, Sev + mel + shMT1. Notice that shMT1 blocked the up‐regulation of PDGFRα, NG2, Sox10, and Olig2 induced by melatonin. (G) Immunostaining of Olig2. N = 6 mice per group. Con, control. Sev, Sevoflurane. mel, melatonin. * p < .05. ** p < .01. *** p < .001. One‐way ANOVA

Article Snippet: MT1 receptor knockout mice were bought from Cyagen Biosciences (Stock No. KOCMP‐17773‐Mtnr1a‐B6N).

Techniques: shRNA, Injection, Expressing, Western Blot, Control, Plasmid Preparation, Immunostaining

Journal: Journal of Pineal Research

Article Title: Melatonin pretreatment alleviates the long‐term synaptic toxicity and dysmyelination induced by neonatal Sevoflurane exposure via MT1 receptor‐mediated Wnt signaling modulation

doi: 10.1111/jpi.12771

Figure Lengend Snippet: Compromising the protective effects of melatonin on Sev‐associated toxicity by MT1 depletion. (A) Experimental design. (B, C) Western blotting and quantification of GluR1, GluR2, and PSD95 in WT and MT1 knockout mice treated with Sev or Sev‐melatonin. (D) Immunostaining of vglut1. Melatonin did not exert protective effects on the expression of GluR1, GluR2, PSD95, and vglut1 in MT1‐KO mice. (E, F) Western blotting and quantification of PDGFRα, Sox10, and Olig2 in WT and melatonin knockout mice treated with Sev or Sev‐melatonin. (G) Immunostaining of Olig2. Melatonin did not exert significant effects on the expression of PDGFRα, Sox10, and Olig2 in MT1 knockout mice. N = 6 mice per group. Con, control. Sev, Sevoflurane. mel, melatonin. WT, wild type. KO, MT1 knockout. * p < .05. ** p < .01. One‐way ANOVA. Bars = 20 μm

Article Snippet: MT1 receptor knockout mice were bought from Cyagen Biosciences (Stock No. KOCMP‐17773‐Mtnr1a‐B6N).

Techniques: Western Blot, Knock-Out, Immunostaining, Expressing, Control

Journal: Journal of Pineal Research

Article Title: Melatonin pretreatment alleviates the long‐term synaptic toxicity and dysmyelination induced by neonatal Sevoflurane exposure via MT1 receptor‐mediated Wnt signaling modulation

doi: 10.1111/jpi.12771

Figure Lengend Snippet: Roles of MT1 knockout in the memory promoting effects of melatonin on Sev‐treated mice. (A) Experimental design. (B, C) Novel object recognition assay. Notice that MT1 knockout mice treated with either Sev or Sev‐melatonin exhibited similar novel object recognition time and similar discrimination index. (D–F) Water Morris maze. MT1 knockout mice treated with either Sev or Sev‐melatonin showed similar escaping time and spent similar time in the target quadrant. N = 8 mice per group. Con, control. Sev, Sevoflurane. mel, melatonin. WT, wild type. KO, MT1 knockout. * p < .05. ** p < .01. One‐way ANOVA

Article Snippet: MT1 receptor knockout mice were bought from Cyagen Biosciences (Stock No. KOCMP‐17773‐Mtnr1a‐B6N).

Techniques: Knock-Out, Control

Journal: Journal of Pineal Research

Article Title: Melatonin pretreatment alleviates the long‐term synaptic toxicity and dysmyelination induced by neonatal Sevoflurane exposure via MT1 receptor‐mediated Wnt signaling modulation

doi: 10.1111/jpi.12771

Figure Lengend Snippet: Long‐term effects of neonatal Sev exposure on MT1 and Wnt signaling in adult hippocampus. (A, B) Western blotting and quantification of p‐CREB, CREB, p‐PKA, and PKA in hippocampus of control, Sev, and Sev‐melatonin‐treated mice at different time points. No changes of p‐CREB, CREB, p‐PKA, and PKA from 7 dps. (C) Heat map of RNA‐seq results of top 20 differential genes in control, Sev‐, and Sev‐melatonin‐treated mice. Notice the changes of SFRP1. (D, E) KEGG enrichment of signaling pathways in Sev‐treated mice and Western blotting of SFRP1. (F) Western blotting and quantification of β‐catenin, p‐β‐catenin (S33/S37/T41) in adult hippocampus of control and Sev‐treated mice. (G) Western blotting and quantification of cytoplasmic and nuclear β‐catenin in adult hippocampus of control and Sev‐treated mice. Notice the decrease in nuclear β‐catenin in Sev‐treated mice. (H, I) qPCR and Western blotting and quantification of Axin2 in adult hippocampus of control and Sev‐treated mice. (J) Double‐immunohistochemistry and quantification of Axin2/NeuN‐ and Axin2/CC1‐positive cells in normal hippocampus. N = 6 mice per group. Con, control. Sev, Sevoflurane. Cyt, cytoplasm. Nuc, nucleus. * p < .05. ** p < .01. Student's t test. One‐way ANOVA. Bars = 50 μm

Article Snippet: MT1 receptor knockout mice were bought from Cyagen Biosciences (Stock No. KOCMP‐17773‐Mtnr1a‐B6N).

Techniques: Western Blot, Control, RNA Sequencing, Protein-Protein interactions, Immunohistochemistry

Journal: Journal of Pineal Research

Article Title: Melatonin pretreatment alleviates the long‐term synaptic toxicity and dysmyelination induced by neonatal Sevoflurane exposure via MT1 receptor‐mediated Wnt signaling modulation

doi: 10.1111/jpi.12771

Figure Lengend Snippet: Interaction between MT1 receptor and β‐catenin. (A, B) Immunostaining of MT1/β‐catenin and MT1/Axin2 in the control and Sev‐treated mice and corresponding quantification at 7 dps. Sev treatment reduced the number of MT1/β‐catenin‐ and MT1/Axin2‐positive cells. (C) Protein CO‐IP assay of MT1/β‐catenin. Sev inhibited the interaction between MT1 and β‐catenin. (D) Double immunostaining of MT1‐C‐Flag and β‐catenin in control and MT1‐C‐Flag transfected cells. (E) Protein CO‐IP assay of MT1‐C/β‐catenin. Notice the co‐localization and interaction of MT1‐C/β‐catenin. (F) Western blotting of Axin2 and SFRP1 in control and MT1‐C overexpressing cells. (G) Western blotting of Axin2 and SFRP1 in control cells, cells treated with MT1‐C‐Flag, and cells treated with combination of MT1‐C‐Flag and shMT1. MT1‐C increased the expression of Axin2 and decreased the expression of SFRP1. (H) Immunostaining of Flag/β‐catenin in MT1‐Flag overexpressing cells under the stimulation of melatonin. Notice the nuclear localization of Flag and β‐catenin upon the stimulation of melatonin. N = 6 mice per group in A–B. N = 3 batches of cells in C–H. * p < .05. ** p < .01, *** p < .001. Student's t test or one‐way ANOVA. Bars = 50 μm (A, B), 20 µm (D), and 10 μm (H)

Article Snippet: MT1 receptor knockout mice were bought from Cyagen Biosciences (Stock No. KOCMP‐17773‐Mtnr1a‐B6N).

Techniques: Immunostaining, Control, Co-Immunoprecipitation Assay, Double Immunostaining, Transfection, Western Blot, Expressing

Journal: Journal of Pineal Research

Article Title: Melatonin pretreatment alleviates the long‐term synaptic toxicity and dysmyelination induced by neonatal Sevoflurane exposure via MT1 receptor‐mediated Wnt signaling modulation

doi: 10.1111/jpi.12771

Figure Lengend Snippet: Long‐term effects of neonatal melatonin pretreatment on the canonical Wnt signaling in adult hippocampus. (A) Protein CO‐IP assay of MT1/β‐catenin in adult hippocampus of mice treated by Sev or Sev‐melatonin at neonatal. Notice that melatonin pretreatment restored the protein interaction between MT1 and β‐catenin. (B, C) Western blotting and quantification of β‐catenin, p‐GSK‐3β(T216), and SFRP1 in adult control, Sev‐treated, and Sev‐melatonin‐treated mice. Melatonin pretreatment reversed the expression of β‐catenin, p‐GSK‐3β(T216), and SFRP1. (D–E) Western blotting and immunostaining of Axin2. In comparison with Sev treatment, Sev‐melatonin increased the expression of Axin2. (F) Western blotting and quantification of Axin2, β‐catenin, and p‐GSK‐3β(T216) in WT mice and MT1 knockout mice (KO) treated with Sev, or Sev‐melatonin. Sev, Sevoflurane. mel, melatonin. WT, wild type. KO, MT1 knockout. N = 6 mice per group. * p < .05. ** p < .01. *** p < .001. One‐way ANOVA. Bar = 50 μm

Article Snippet: MT1 receptor knockout mice were bought from Cyagen Biosciences (Stock No. KOCMP‐17773‐Mtnr1a‐B6N).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Control, Expressing, Immunostaining, Comparison, Knock-Out

Journal: Journal of Pineal Research

Article Title: Melatonin pretreatment alleviates the long‐term synaptic toxicity and dysmyelination induced by neonatal Sevoflurane exposure via MT1 receptor‐mediated Wnt signaling modulation

doi: 10.1111/jpi.12771

Figure Lengend Snippet: Schematic drawing of the major finding. (A) Under normal condition, hippocampal neurons and oligodendrocytes express MT1 receptor. MT1 co‐localizes with β‐catenin at membrane and binds to β‐catenin with its C‐terminal. (B) Left panel: upon Sev stimulation, β‐catenin dissociates from MT1, leading to up‐regulation of SFRP1 and suppression Wnt signaling. Right panel: at the presence of melatonin, MT1/β‐catenin interaction is strengthened; thus, Wnt signaling is enhanced and SFRP1 lowered

Article Snippet: MT1 receptor knockout mice were bought from Cyagen Biosciences (Stock No. KOCMP‐17773‐Mtnr1a‐B6N).

Techniques: Membrane

Journal: PLoS ONE

Article Title: Recombinant Human Melatonin Receptor MT1 Isolated in Mixed Detergents Shows Pharmacology Similar to That in Mammalian Cell Membranes

doi: 10.1371/journal.pone.0100616

Figure Lengend Snippet: Expression levels of the human MT1 receptor were assessed using a [ 3 H]-melatonin ligand binding assay. N: number of independent experiments. Schematic representations of the evaluated expression vectors use the following abbreviations: MT1, human MT1 receptor; His, 10-histidine tag; attB1 and attB2, recombination sites of the Gateway system; Tev, tobacco etch virus protease cleavage site; Trx protein, thioredoxin protein; α-F, sequence signal of the Saccharomyces cerevisiae α-Factor; Flag, flag-epitope tag; Biotin, biotinylation domain from Propionibacterium shermanii ; Gαi1, αi1 subunit of G protein; ss, signal sequence from influenza hemagglutinin gene; YFP, yellow fluorescent protein; and 2-Strep, double Strep tag.

Article Snippet: For the cell-free/liposome system, the Synthelis Company produced the MT1 receptor from the two dedicated plasmids, using E. coli cell lysate with preformed liposomes in the presence or absence of MT1 ligands.

Techniques: Expressing, Ligand Binding Assay, Virus, Sequencing, FLAG-tag, Strep-tag

Journal: PLoS ONE

Article Title: Recombinant Human Melatonin Receptor MT1 Isolated in Mixed Detergents Shows Pharmacology Similar to That in Mammalian Cell Membranes

doi: 10.1371/journal.pone.0100616

Figure Lengend Snippet: P. Pastoris membranes were solubilized in the presence of a panel of detergent concentrations indicated on the figures. Solubilized proteins were then partially purified on Ni-NTA Spin columns, and finally analyzed using a [ 3 H]-melatonin binding assay ( A ) and analytical size exclusion chromatography (SEC) ( B and C ). A , MB: P. pastoris membranes expressing MT1 receptor. B and C , Protein absorbance profiles measured at 280 nm; white triangles: MT1 oligomers; black triangles: MT1 monomers.

Article Snippet: For the cell-free/liposome system, the Synthelis Company produced the MT1 receptor from the two dedicated plasmids, using E. coli cell lysate with preformed liposomes in the presence or absence of MT1 ligands.

Techniques: Purification, Binding Assay, Size-exclusion Chromatography, Expressing

Journal: PLoS ONE

Article Title: Recombinant Human Melatonin Receptor MT1 Isolated in Mixed Detergents Shows Pharmacology Similar to That in Mammalian Cell Membranes

doi: 10.1371/journal.pone.0100616

Figure Lengend Snippet: P. pastoris membranes were solubilized with 1% CHAPS ( A ) or 0.25% Fos14 ( B ), and purified in the presence of the indicated concentration of detergents using a two-step purification approach consisting of anti-flag affinity chromatography followed by size exclusion chromatography. 1 and 5 : Size exclusion chromatography profile. Red arrows indicate SEC elution fractions F17, corresponding to MT1 oligomers, and F22, corresponding to MT1 monomers. 2 and 6 : SDS-PAGE of SEC elution fractions F17 and F22 colored with Coomassie Blue (left) and revealed by anti-Flag immunodetection (right). 3 and 7 : Saturation ligand binding experiments with [ 3 H]-melatonin on SEC elution fraction F17 for CHAPS and F17 or F22 for Fos14. 4 and 8 : Negative staining electron microscopy on SEC elution fractions F17 for CHAPS and F22 for Fos14.

Article Snippet: For the cell-free/liposome system, the Synthelis Company produced the MT1 receptor from the two dedicated plasmids, using E. coli cell lysate with preformed liposomes in the presence or absence of MT1 ligands.

Techniques: Purification, Concentration Assay, Affinity Chromatography, Size-exclusion Chromatography, SDS Page, Immunodetection, Ligand Binding Assay, Negative Staining, Electron Microscopy

Journal: PLoS ONE

Article Title: Recombinant Human Melatonin Receptor MT1 Isolated in Mixed Detergents Shows Pharmacology Similar to That in Mammalian Cell Membranes

doi: 10.1371/journal.pone.0100616

Figure Lengend Snippet: Comparison of binding affinities (Ki) of MT1 receptors in CHO and Pichia pastoris (P.p.) membranes, and purified in a 0.1% Fos14/0.1% CHAPS mixture.

Article Snippet: For the cell-free/liposome system, the Synthelis Company produced the MT1 receptor from the two dedicated plasmids, using E. coli cell lysate with preformed liposomes in the presence or absence of MT1 ligands.

Techniques: Comparison, Binding Assay, Purification

Journal: PLoS ONE

Article Title: Recombinant Human Melatonin Receptor MT1 Isolated in Mixed Detergents Shows Pharmacology Similar to That in Mammalian Cell Membranes

doi: 10.1371/journal.pone.0100616

Figure Lengend Snippet: A , pKi correlation of MT1 in P. pastoris membranes vs. MT1 in CHO membranes. B , pKi correlation of MT1 purified in Fos14/CHAPS vs. MT1 in CHO membranes. C , pKi correlation of MT1 in P. pastoris membranes vs. MT1 purified in Fos14/CHAPS. Processed data are presented in . Pearson's correlation analyses revealed r coefficients of 0.908 ( p <0.0001, n = 4), 0.840 ( p <0.0001, n = 4), and 0.840 ( p <0.0001, n = 4) for A , B , and C , respectively.

Article Snippet: For the cell-free/liposome system, the Synthelis Company produced the MT1 receptor from the two dedicated plasmids, using E. coli cell lysate with preformed liposomes in the presence or absence of MT1 ligands.

Techniques: Purification