Journal: Nature neuroscience

Article Title: NCOR1/2 loss of function impairs memory through a novel GABAergic hypothalamus–CA3 projection

doi: 10.1038/s41593-018-0311-1

Figure Lengend Snippet: . (a) Patients carrying copy number variants (CNV) or single nucleotide variants (SNV) in NCOR1 , NCOR2 , or HDAC3 . Genomic coordinates are shown in hg19. DDD_SNV, single nucleotide variants retrieved from Deciphering Developmental Disorders (DDD) website (United Kingdom); kb, kilobase. (b-d) Schematic representations for the deletions and point mutations affecting NCOR1 , NCOR2 , or HDAC3 , respectively, observed in patients with neurodevelopmental disorders. The locations of deletions are depicted in red, and the point mutations in pink. (e) Western blot of HEK-293 cells transfected with plasmids expressing wild-type (WT) HDAC3 with or without mutant L266S. The experiment was repeated independently once with similar results. The blot images have been cropped. (f) Fluorescence-based HDAC enzyme assay after anti-HDAC3 immunoprecipitates from cell lysates overexpressing the indicated HDAC3 proteins. Box plots center line, median; box limits, upper and lower quartiles; whiskers, minimal and maximum values. Data were analyzed by two-tailed unpaired t test. n=4 biological independent samples for each group. (g) Western blot of HEK-293 cells transfected with plasmids expressing WT HDAC3, WT NCOR1, with or without the NCOR1 deletion mutant (Del). The experiment was repeated independently once with similar results. Data were analyzed by two-tailed unpaired t test. n=3 biological independent samples for each group. The blot images have been cropped. (h) Chromatin immunoprecipitation (ChIP) with anti-HDAC3 antibodies followed by qPCR using primers targeting promoters of the indicated genes ARNTL and CDKN1A . RPLP0 serves as a negative control. Data is expressed as mean ± S.E.M. For detailed statistics results, see . * P ≤ 0.05 is set as significance.

Article Snippet: For western blot, immunoprecipitates and total tissue lysates were resolved by Tris-glycine SDS-PAGE, transferred to PVDF membranes, and blotted with antibodies against NCOR1 (lab-made) , TBLR1 (IMGENEX, IMG591), and HDAC3 (Abcam 7030).

Techniques: Western Blot, Transfection, Expressing, Mutagenesis, Fluorescence, Enzymatic Assay, Two Tailed Test, Chromatin Immunoprecipitation, Negative Control

Journal: Hepatology (Baltimore, Md.)

Article Title: Phosphorylation of the Nuclear Receptor Co-repressor 1 by Protein Kinase B (PKB/Akt) Switches its Co-repressor Targets in the Liver

doi: 10.1002/hep.27907

Figure Lengend Snippet: (A) Heat maps displaying expression values of each gene at the indicated Zeitgeber time and Pol II-density profiles of Scd1, Cpt1a, and Cox7a1 showing diurnal expression of lipogenic, ketogenic and OxPhos genes in liver (GSE35790). (B) NCoR1 phosphorylation by insulin treatment in HEK293T cells. Cells were transfected with Flag-tagged mouse NCoR1 (1 µg/well of 6-well plate). After 48 hours, cells were treated with insulin (100nM) for the indicated times. (C) Schematic presentation of putative mouse NCoR1 phosphorylation sites by Akt. Positions are numbered according to NCBI Reference Sequence: NP_001239242.1. RD, repression domain; SANT, the SANT-like domains; DAD, deacetylase activation domain; HID, histone interaction domain; RIDs, nuclear receptor interacting domains. AA sequences indicates (Arg-Xaa-)Arg-Xaa-Xaa-Ser/Thr motifs that have been reported to be phosphorylated by Akt/PKB in sequence 1 and 2. (D) In vitro kinase activity of Akt1 toward two short synthetic GST-tagged peptides, each containing 1 of the 2 putative Akt phosphorylation sites of NCoR1, was analyzed by autoradiography. (E) In vitro kinase activity of Akt1 toward a synthetic GST-tagged peptide containing 1460 serine residue of NCoR1. Coomassie stain was performed to visualize the short synthetic peptides, which were used for subsequent Mass spectrometry. (F) Diagrams showing the phosphorylation of 1460 serine residue of NCoR1 by LC-MS/MS. (G) In vitro kinase activity of Akt1 toward two short synthetic GST-tagged peptides, containing either the wild-type 1460 serine residue or mutant 1460 alanine residue of NCoR1. (H) Sequence alignment of the NCoR1 phosphorylation site in various vertebrate species. All immunoblots are representative of at least three independent experiments.

Article Snippet: But, polyclonal rabbit antibody to detect NCoR1, which is specifically phosphorylated on 1460 serine (Anti-pS1460 NCoR1 Ab) was generated in Young In Frontier Co., Ltd (Seoul, South Korea).

Techniques: Expressing, Transfection, Sequencing, Histone Deacetylase Assay, Activation Assay, In Vitro, Activity Assay, Autoradiography, Staining, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Mutagenesis, Western Blot

Journal: Hepatology (Baltimore, Md.)

Article Title: Phosphorylation of the Nuclear Receptor Co-repressor 1 by Protein Kinase B (PKB/Akt) Switches its Co-repressor Targets in the Liver

doi: 10.1002/hep.27907

Figure Lengend Snippet: (A) Co-immunoprecipitation assay using wild-type Flag-NCoR1, Flag-S1460E NCoR1 (SE), Flag-S1460A NCoR1 (SA) and HA-LXRα in HEK293T cells. Cells were transfected with indicated plasmids (1 µg/well per 6-well plate). After 48 hours, cell lysates were immunoprecipitated with anti-Flag antibody. (B and C) Similar co-immunoprecipitation assays using the same NCoR1 constructs as in (A) and HA-PPARα (B) and HA-ERRα (C). (D-F) NCoR1 recruitment to the LXRE (D), PPRE (E) and ERRE (F) sites of the mouse Srebp1c, Cpt1a and Sdhb promoters determined by ChIP in AML12 cells and quantitative RT-PCR (qRT-PCR) analysis of the mRNA levels of Srebp1c (D), Cpt1a (E) and Sdhb (F) according to the sequence composition of NCoR1 phosphorylation site (each group, n>5). mRNA levels were analyzed in AML12 cells transfected with indicated NCoR1 plasmids (each group, n>7) (G) Co-immunoprecipitation assay using wild-type Flag-NCoR1, Flag-S1460A NCoR1 (SA) and HA-LXRα (1 µg/well per 6-well plate) in AML12 cells with or without GW3965 (1 µM) as indicated. (H) NCoR1 recruitment to the LXRE of the mouse Srebp1c, promoters determined by ChIP and quantitative RT-PCR (qRT-PCR) analysis of the mRNA levels of Srebp1c in AML12 cells according to the sequence composition of the S1460 NCoR1 phosphorylation site in the absence or presence of insulin (100 nM) for 6 h with or without LY294002 (30 µM) given 30 min before insulin treatment (each group, n>5). (I) qRT-PCR analysis to evaluate mRNA levels of Srebp1c and Fasn in primary hepatocytes from NCoR1L2/L2 mice infected with either an Ad-LacZ or Ad-Cre adenovirus grown in high glucose (25 mM) medium with or without insulin treatment (100nM) for 6 h. All immunoblots are representative of at least three independent experiments. Data are mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: But, polyclonal rabbit antibody to detect NCoR1, which is specifically phosphorylated on 1460 serine (Anti-pS1460 NCoR1 Ab) was generated in Young In Frontier Co., Ltd (Seoul, South Korea).

Techniques: Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Construct, Quantitative RT-PCR, Sequencing, Infection, Western Blot

Journal: Hepatology (Baltimore, Md.)

Article Title: Phosphorylation of the Nuclear Receptor Co-repressor 1 by Protein Kinase B (PKB/Akt) Switches its Co-repressor Targets in the Liver

doi: 10.1002/hep.27907

Figure Lengend Snippet: Acute NCoR1 deletion results in induction of lipogenic and OxPhos genes. (A) qRT-PCR analysis to evaluate mRNA levels of Ndufb5, Sdhb, Uqcrfs1, Cox5a, Atp5g1 and Ncor1 in NCoR1L2/L2 MEF cells infected with either an Ad-LacZ or Ad-Cre adenovirus grown in high glucose (25 mM) medium. (B) Western blot analysis showing Atp5a, Uqcrc2, Mtco1, Sdhb and Ndufb8 levels in NCoR1L2/L2 MEFs infected during 72-hr with either an Ad-LacZ or Ad-Cre adenovirus. (C) BN-PAGE of OXPHOS complexes in mitochondria isolated from NCoR1L2/L2 MEFs infected with either an Ad-LacZ or Ad-Cre adenovirus. (D) Oxygen consumption rate (OCR) was measured in NCoR1L2/L2 MEFs infected with either an Ad-LacZ or Ad-Cre adenovirus grown in high glucose (25 mM) medium (n=6/group). Data are mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001. (E) qRT-PCR analysis to evaluate mRNA levels of Ndufb5, Sdhb, Uqcrfs1, Cox5a, Atp5g1, Me1, G6pdx, Scd1, Gpam, Fasn, Plin2 and Ncor1 in primary hepatocytes from NCoR1L2/L2 mice infected with either an Ad-LacZ or Ad-Cre adenovirus grown in high glucose (25 mM) medium.

Article Snippet: But, polyclonal rabbit antibody to detect NCoR1, which is specifically phosphorylated on 1460 serine (Anti-pS1460 NCoR1 Ab) was generated in Young In Frontier Co., Ltd (Seoul, South Korea).

Techniques: Quantitative RT-PCR, Infection, Western Blot, Isolation

Journal: Hepatology (Baltimore, Md.)

Article Title: Phosphorylation of the Nuclear Receptor Co-repressor 1 by Protein Kinase B (PKB/Akt) Switches its Co-repressor Targets in the Liver

doi: 10.1002/hep.27907

Figure Lengend Snippet: (A) mRNA levels of Me1, Fasn, Elovl6, Srebp1, Srebp2, Acox1, mCad, Cpt1a, Esrra, Ndufb5 and Sdhb were evaluated by qRT-PCR in livers of NCoR1hep+/+ and NCoR1hep−/− mice fasted for 24-hr (n > 7/group). All data shown as mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001. (B) BN-PAGE of OXPHOS complexes in mitochondria isolated from liver of 25-wk old male NCoR1hep+/+ and NCoR1hep−/− mice either fed or fasted for 24-hr. Upper panel is a representative result developed by chromogen-based detection technique to visualize supercomplexes (SC), complex I, V and III. The lower panel shows HRP-based detection to evaluate complex II amount. (C) Western blot analysis indicating that insulin increases pS1460 NCoR1 in livers from 25-wk old NCoR1hep+/+ and NCoR1hep−/− male chow fed mice. Animals were injected with either PBS or insulin (0.5 U/kg body weight) at 10 min before sacrificing. (D) Western blot analysis showing that PI3K inhibition attenuates the insulin-induced increases of pS1460 NCoR1 in livers from 25-wk old 4-hr fasted C57BL/6J male chow fed mice. After 4 hrs of fasting, animals were injected with either PBS or insulin (0.5 U/kg body weight) for 10 min or 30 min with or without LY294002 (40 mg/kg) which was given 60 min before sacrifice. (E) pS1460 NCoR1 levels in livers from 25-wk old C57BL/6J male mice fed with either chow or high fat diet for 12-wk. (F) pS1460 NCoR1 levels in livers from 12-wk old male db/db mice. Animals in (E) and (F) were fasted for 4-hr. The experimental protocol is similar to that used in (C). All immunoblots are representative of at least three independent experiments. (G) Working model, summarizing how NCoR1 may control the fasting-feeding transitions in liver in a phosphorylation dependent manner.

Article Snippet: But, polyclonal rabbit antibody to detect NCoR1, which is specifically phosphorylated on 1460 serine (Anti-pS1460 NCoR1 Ab) was generated in Young In Frontier Co., Ltd (Seoul, South Korea).

Techniques: Quantitative RT-PCR, Isolation, Western Blot, Injection, Inhibition