Journal: Nature chemical biology

Article Title: Identification and characterization of PPARα ligands in the hippocampus

doi: 10.1038/nchembio.2204

Figure Lengend Snippet: GC-MS analyses of chloroform- (A & B) and acetonitrile- (C) reconstituted nuclear extracts of WT hippocampus after pulling down with GST-PPARα-LBD. Similar GC-MS analyses were performed in chloroform (D) and acetonitrile (E) reconstituted nuclear extracts after pulling down with GST-PPARβ-LBD. F) The immunoblot analyses of eluate collected from glutathione column probed with anti-GST antibody (upper panel), and anti-PPARα or anti-PPARβ antibodies (lower panel). Histone 3 (H3) immunoblot was performed in the nuclear lysate (input) to show the purity of the nuclear extract (middle panel). For raw uncut blots, please see . GC-MS analyses of the chloroform-extracted nuclear fraction of lenti-vector- (G) and lenti-PPARα- (H) transduced Ppara -null hippocampal neurons. I) Neuronal extracts infected with lenti-vector and lenti-PPARα were analyzed for PPARα and then normalized with actin. For raw uncut blots, please see . Results were confirmed by three independent experiments.

Article Snippet: The mouse PPARα ORF cloned in the pCMV6-AC-GFP vector (cat # MG 227641) was purchased from Origene.

Techniques: Gas Chromatography-Mass Spectrometry, Western Blot, Plasmid Preparation, Infection

Journal: Nature chemical biology

Article Title: Identification and characterization of PPARα ligands in the hippocampus

doi: 10.1038/nchembio.2204

Figure Lengend Snippet: TR-FRET analyses were performed and fitted curves are shown for OCT (A), HEX (B) and HMB (C). Dose response curves were plotted as a ratio of fluorescence response with increasing doses of agonists. Graph-pad prism 7 software was used to draw a sigmoidal curve-fit. Respective EC50 (4.838 μM for OCT, 5.264 μM for HEX and 35.85 μM for HMB) and hill slope (9.01 for OCT, 8.982 for HEX and 6.747 for HMB) values were calculated based on sigmoidal curve-fit equation: Y=Bottom + (X Hillslope )*(Top-Bottom)/(X Hillslope + EC50 Hillslope ). Thermal-shift assay of OCT (D), HEX (E) and HMB (F) was performed using 5 μM OCT, 5 μM HEX and 25 μM HMB as described under the Materials and Method section. Equation for full-length protein only: y = 50 = - 0.0652 x 3 + 9.053 x 2 - 408.09 x + 6012.7 ; x = 45.96321 Equation for full length protein with OCT: y = 50 = - 0.0002 x 5 + 0.052 x 4 - 5.1349 x 3 + 250.52 x 2 - 6041.9 x + 57653 ; x = 59.6128 Equation for full length protein with HEX: y = 0.0074 x 3 - 0.8528 x 2 + 31.967 x - 389.74 ; x = 59.2835 Equation for full length protein with HMB: y = - 0.06529 x 3 + 9.053 x 2 - 408.09 x + 6012.7 ; x = 58.494798 Ribbon representations of superposed structures of PPARα ligand binding pocket along with its ligands OCT (G), HEX (H) and HMB (I) are shown. Blue dotted lines represent potential hydrogen bonds. Results are confirmed by three independent experiments.

Article Snippet: The mouse PPARα ORF cloned in the pCMV6-AC-GFP vector (cat # MG 227641) was purchased from Origene.

Techniques: Fluorescence, Software, Thermal Shift Assay, Ligand Binding Assay

Journal: Nature chemical biology

Article Title: Identification and characterization of PPARα ligands in the hippocampus

doi: 10.1038/nchembio.2204

Figure Lengend Snippet: Ribbon representations of superposed structures of Y464D/Y314D-PPARα ligand binding pocket along with OCT (A), HEX (B) and HMB (C). Thermal shift assays of FL-PPARα (D) and Y314D/Y464D-PPARα (E) proteins. Tm represents the melting temperature. F) Thermal shift assay for Y464D-PPARα alone and together with three ligands. GC-MS analyses in GFP-affinity purified extracts of Ppara -null hippocampal neurons transduced with lentivirions containing GFP-Y314D-Ppara (G), GFP-Y464D-Ppara (H), and GFP-Y314D/Y464D-Ppara (I).

Article Snippet: The mouse PPARα ORF cloned in the pCMV6-AC-GFP vector (cat # MG 227641) was purchased from Origene.

Techniques: Ligand Binding Assay, Thermal Shift Assay, Gas Chromatography-Mass Spectrometry, Affinity Purification, Transduction

Journal: Foods

Article Title: Sea Buckthorn Pericarp Flavonoids Improve Diet-Induced Hyperlipidemia via Coordinated Modulation of Hepatic Lipid Metabolism and Gut Microbiota

doi: 10.3390/foods15061049

Figure Lengend Snippet: TFSP modulates the expression of ACC, FAS, CPT-1α, PPARα and ATGL proteins in the livers of HFD mice. n = 6 per group. Data are presented as mean ± SD. Different letters above bars indicate statistically significant differences ( p < 0.05) by one-way ANOVA followed by LSD post hoc test.

Article Snippet: Peroxisome proliferator-activated receptor alpha (PPARα) primary antibody (Cat. No. A00600-2), carnitine palmitoyltransferase-1 alpha (CPT-1α) primary antibody (Cat. No. A00917-3), acetyl-CoA carboxylase (ACC) primary antibody (Cat. No. M01802-2), fatty acid synthase (FAS) primary antibody (Cat. No. BA0484), adipose triglyceride lipase (ATGL) primary antibody (Cat. No. A01800-1), and GAPDH primary antibody (Cat. No. BM1623) were obtained from BOSTER Biological Technology Co., Ltd. (Wuhan, China).

Techniques: Expressing

Journal: Nature Chemical Biology

Article Title: Identification and characterization of PPARα ligands in the hippocampus

doi: 10.1038/nchembio.2204

Figure Lengend Snippet: Figure 1 | PPAR is critical in regulating the expression of synaptic molecules in hippocampal neurons. (a) Heat map of PCR-based microarray analysis of plasticity-associated genes in the hippocampus of WT and Ppara-null (KO) mice. n = 3 mice per group. (b) Number of genes inhibited (28; purple), stimulated (34; gray) and unchanged (22; overlapped) in Ppara-null hippocampus. (c) Real-time PCR analyses of Arc, Creb, Grin2a, Grin2b, and Gria1 mRNA expression in Ppara-null mice. Results are mean ± s.e.m. of 3 mice. (d) MAP-2- and PSD-95-specific immunostaining in hippocampal tissue of 6- to 8-week-old WT (n = 3) and Ppara-null (n = 3) mice. Representative image from CA1 region of the hippocampus. Scale bars, 10 μm. (e) Magnified view of region enclosed in the box is in d. Scale bars, 10 μm. Results represent analysis of 3 hippocampal sections each from 3 mice per group. (f,g) Western blot (f) and densitometric analysis (g) of NR-2A, GluR1, PSD-95, Arc, and CREB expression (normalized to actin) in hippocampal tissue of WT (n = 3) and Ppara- null (n = 3) mice. Full blots are shown in Supplementary Figure 13a. Results are mean ± s.e.m. of 3 mice. *P < 0.001 vs. WT (one-way ANOVA (single factor: genotype) coupled with t-test).

Article Snippet: The mouse Ppara ORF cloned in the pCMV6-AC-GFP vector was purchased from Origene (MG 227641).

Techniques: Expressing, Microarray, Real-time Polymerase Chain Reaction, Immunostaining, Western Blot

Journal: Nature Chemical Biology

Article Title: Identification and characterization of PPARα ligands in the hippocampus

doi: 10.1038/nchembio.2204

Figure Lengend Snippet: Figure 2 | Identification of endogenous ligands of PPAR in the mouse hippocampus. (a–c) GC-MS analyses of chloroform-reconstituted (a,b) and acetonitrile-reconstituted (c) nuclear extracts of WT hippocampus after pulling down with GST-tagged PPARα LBD. (d,e) GC-MS analyses similar to those in a–c performed in chloroform-reconstituted (d) and acetonitrile-reconstituted (e) nuclear extracts after pulling down with GST-tagged PPARβ LBD. (f) Immunoprecipitation (IP) and immunoblot (IB) analyses of eluate collected from glutathione column probed with anti-GST antibody (top) and anti-PPARα or anti-PPARβ antibodies (bottom). Histone 3 (H3)-specific immunoblot was performed on nuclear lysate (input) to show the purity of the nuclear extract (middle). Full blots are shown in Supplementary Figure 13b. (g,h) GC-MS analyses of the chloroform-extracted nuclear fraction of Ppara-null hippocampal neurons transduced with lenti-vector (g) or lenti-FL-Pparα (h). (i) PPARα expression in neuronal extracts infected with lenti-vector (vector) or lenti-FL-Pparα (Ppara); results are normalized to actin. Full blots are shown in Supplementary Figure 13c. Results were confirmed in 3 independent experiments (a–i).

Article Snippet: The mouse Ppara ORF cloned in the pCMV6-AC-GFP vector was purchased from Origene (MG 227641).

Techniques: Gas Chromatography-Mass Spectrometry, Immunoprecipitation, Western Blot, Transduction, Plasmid Preparation, Expressing, Infection

Journal: Nature Chemical Biology

Article Title: Identification and characterization of PPARα ligands in the hippocampus

doi: 10.1038/nchembio.2204

Figure Lengend Snippet: Figure 4 | Interaction between ligands and PPARa at the molecular level. (a–c) Ribbon representations of superimposed structures of Y464D/Y314D-PPARα LBD with OCT (a), HEX (b), and HMB (c). (d,e) Thermal shift assays of GFP-FL-PPARα (d) and GFP-Y314D/Y464D-PPARα (e). (f) Thermal shift assays for GFP-Y464D-PPARα alone or with OCT, HEX, or HMB. (g–i) GC-MS analyses in GFP-affinity purified extracts of Ppara-null hippocampal neurons transduced with lenti-GFP-Y314D-Ppara (g), lenti-GFP-Y464D-Ppara (h), and lenti-GFP-Y314D/Y464D-Ppara (i). Relative (rel.) fluorescence values were calculated considering maximum fluorescence response as 100%. Results (a–i) were confirmed by 3 independent experiments with 3 samples per experiment.

Article Snippet: The mouse Ppara ORF cloned in the pCMV6-AC-GFP vector was purchased from Origene (MG 227641).

Techniques: Gas Chromatography-Mass Spectrometry, Affinity Purification, Transduction, Fluorescence

Journal: Nature Chemical Biology

Article Title: Identification and characterization of PPARα ligands in the hippocampus

doi: 10.1038/nchembio.2204

Figure Lengend Snippet: Figure 6 | Effect of PPAR ligands on morphological plasticity and calcium oscillation in hippocampal neurons. (a–e) Spine density in Ppara-null hippocampal neurons transduced with lenti-vector (vector), lenti-FL-Ppara (FL Ppara), or lenti-Y464D-Ppara (Y464D) for 48 h followed by treatment with vehicle (DMSO) (a), OCT (b), HEX (c), HMB (d), or WY14643 (e) for 24 h. Boxes show the whole hippocampal neuron. Scale bars, 20 μm. (f–m) AMPA-driven (f–i) or NMDA-driven (j–m) calcium influx in OCT-, HEX-, and HMB-treated Ppara-null hippocampal neurons transduced with lenti-FL-Ppara (f,j), lenti-Y314D-Ppara (g,k), lenti-Y464D- Ppara (h,l), or lenti-Y314D/Y464D-Ppara (i,m). Neurons were treated with 50 μM NMDA receptor antagonist N20C (f–i) or Naspm-HCl (j–m) to inhibit passive calcium flow through the receptors. Results are mean of 3 independent experiments analyzing 3 samples each.

Article Snippet: The mouse Ppara ORF cloned in the pCMV6-AC-GFP vector was purchased from Origene (MG 227641).

Techniques: Transduction, Plasmid Preparation