Journal: Molecules and Cells

Article Title: Regulation of Hepatic Gluconeogenesis by Nuclear Receptor Coactivator 6

doi: 10.14348/molcells.2022.2222

Figure Lengend Snippet: (A) Schematic representation of the liver-specific knockout strategy used for the Ncoa6 gene, based on the Cre-LoxP system. Blue arrows denote the specific primer binding sites for genotyping. (B-D) The liver specific knockout of Ncoa6 was confirmed at the level of genomic DNA (gDNA), mRNA and protein. gDNA and mRNA were isolated from the liver, muscle, kidney and white adipose tissue (WAT) of Ncoa6 fl/fl /Cre +/- (LKO) and Ncoa6 fl/fl /Cre -/- (F/F) mice. The Ncoa6 gDNA levels were determined by conventional PCR (B). The Ncoa6 mRNA and NCOA6 protein levels were determined by qRT-PCR (C; n = 4) and western blotting (D; n = 3), respectively. mRNA data are presented as the mean ± SEM; *** P < 0.001 by t -test comparisons of the genotypes.

Article Snippet: After membrane blocking with 5% skim milk, the membrane was probed with primary antibodies against NCOA6 (Cat. No. NB200-335; Novus Biologicals, USA), PCK1 (Cat. No. sc-32879; Santa Cruz Biotechnology, USA), CREB (Cat. No. 9197; Cell Signaling Technology, USA), p-CREB (Cat. No. 9198S; Cell Signaling Technology), or α-tubulin (Cat. No. T9026; Sigma-Aldrich).

Techniques: Knock-Out, Binding Assay, Isolation, Quantitative RT-PCR, Western Blot

Journal: Molecules and Cells

Article Title: Regulation of Hepatic Gluconeogenesis by Nuclear Receptor Coactivator 6

doi: 10.14348/molcells.2022.2222

Figure Lengend Snippet: (A) The blood glucose levels were measured in Ncoa6 LKO (n = 8) and WT (F/F, n = 9) mice after 16 h fasting. (B) Improved glucose tolerance in the Ncoa6 LKO mice. Intraperitoneal glucose tolerance tests were performed in 10-week-old mice (n = 9 for F/F, n = 8 for LKO) after a 16 h fast. (C) The serum insulin levels were measured in Ncoa6 LKO (n = 6 for fed, n = 7 for fasted) and WT mice (n = 7 for both fed and fasted) before and after a 16 h fast. (D) Enhanced insulin sensitivity in Ncoa6 LKO mice. Intraperitoneal insulin tolerance tests were performed in 10-week-old Ncoa6 LKO (n = 13) and WT mice (n = 10) after a 6 h fast. (E) Pyruvate tolerance was determined in 10-week-old Ncoa6 LKO (n = 11) and WT (n = 10) mice via an intraperitoneal injection with pyruvate (2 g/kg body weight) after overnight fasting for 16 h. AUC values for GTT, ITT, and PTT were calculated and are presented on the right of each graph. Data are presented as the mean ± SEM; * P < 0.05, ** P < 0.01 by t -test comparisons of the genotypes. WT, wild type; GTT, glucose tolerance test; ITT, insulin tolerance test; PTT, pyruvate tolerance test.

Article Snippet: After membrane blocking with 5% skim milk, the membrane was probed with primary antibodies against NCOA6 (Cat. No. NB200-335; Novus Biologicals, USA), PCK1 (Cat. No. sc-32879; Santa Cruz Biotechnology, USA), CREB (Cat. No. 9197; Cell Signaling Technology, USA), p-CREB (Cat. No. 9198S; Cell Signaling Technology), or α-tubulin (Cat. No. T9026; Sigma-Aldrich).

Techniques: Injection

Journal: Molecules and Cells

Article Title: Regulation of Hepatic Gluconeogenesis by Nuclear Receptor Coactivator 6

doi: 10.14348/molcells.2022.2222

Figure Lengend Snippet: (A) Decreased induction of gluconeogenic gene transcripts in the Ncoa6 LKO liver following fasting. Liver RNAs were isolated from Ncoa6 LKO and WT mice after a 24 h fast (n = 6 for each group). The mRNA levels of Pc , Pck1 , and G6pc were determined by real-time qRT-PCR. (B) Attenuated induction of PCK protein in the Ncoa6 LKO liver after fasting. Protein expression was analyzed in the liver of ad libitum fed mice and 16 h fasted mice (n = 7 for each group) by western blot analysis. Intensities of the protein bands were measured using the ImageJ program. (C) Glucagon tolerance of Ncoa6 LKO mice (n = 10 for F/F, n = 14 for LKO). Ten-week-old mice were injected intraperitoneally with glucagon (15 µg/kg) after 6 h of fasting. Blood samples were prepared at 0, 30, 60, 90, and 120 min after the glucagon injection. AUC values are shown on the right. (D) Effects of NCOA6 on the glucose production level in primary hepatocytes. Glucose production was compared between Ncoa6 LKO and WT mouse primary hepatocytes after a 4 h forskolin treatment using glucose free media supplemented with lactate and sodium pyruvate (n = 4 independent experiments). (E) Alteration of Pck1 and G6pc transcript induction in Ncoa6 LKO primary hepatocytes following a 4 h treatment of these cells with forskolin (n = 5 independent experiments). Transcript levels were analyzed by real-time qRT-PCR. Data are presented as the mean ± SEM; ** P < 0.01, *** P < 0.001, determined using one-way ANOVA. Fsk, forskolin.

Article Snippet: After membrane blocking with 5% skim milk, the membrane was probed with primary antibodies against NCOA6 (Cat. No. NB200-335; Novus Biologicals, USA), PCK1 (Cat. No. sc-32879; Santa Cruz Biotechnology, USA), CREB (Cat. No. 9197; Cell Signaling Technology, USA), p-CREB (Cat. No. 9198S; Cell Signaling Technology), or α-tubulin (Cat. No. T9026; Sigma-Aldrich).

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

Journal: Molecules and Cells

Article Title: Regulation of Hepatic Gluconeogenesis by Nuclear Receptor Coactivator 6

doi: 10.14348/molcells.2022.2222

Figure Lengend Snippet: (A) Serum glucagon levels in the Ncoa6 LKO (n = 6 for fed, n = 7 for fasted) and WT mice (n = 7 for both fed and fasted) before and after 16 h of fasting. (B) mRNA levels of gluconeogenic transcriptional factors in the liver of Ncoa6 LKO and WT mice in a fed or 24 h fasted state (n = 6 for each group). Hepatic mRNAs were analyzed by real-time qRT-PCR. (C) Transcript levels of the CREB target gene Nr4a1 and FOXO1 target gene Igfbp1 in the primary hepatocytes of Ncoa6 LKO (n = 4) and WT mice (n = 3). (D) Induced phosphorylation of CREB by 16 h of fasting in the livers of Ncoa6 LKO and WT mice (n = 3 for each group). (E) Forskolin-induced phosphorylation of CREB in primary hepatocytes of Ncoa6 LKO and WT mice. Protein levels were determined by western blot analyses using anti-CREB, anti-pCREB (Ser133) or anti-tubulin antibodies (n = 5 independent experiments). The intensities of the protein bands were measured using the ImageJ program. Data are presented as the mean ± SEM; *** P < 0.001, determined using one-way ANOVA. Fsk, forskolin.

Article Snippet: After membrane blocking with 5% skim milk, the membrane was probed with primary antibodies against NCOA6 (Cat. No. NB200-335; Novus Biologicals, USA), PCK1 (Cat. No. sc-32879; Santa Cruz Biotechnology, USA), CREB (Cat. No. 9197; Cell Signaling Technology, USA), p-CREB (Cat. No. 9198S; Cell Signaling Technology), or α-tubulin (Cat. No. T9026; Sigma-Aldrich).

Techniques: Quantitative RT-PCR, Phospho-proteomics, Western Blot

Journal: Molecules and Cells

Article Title: Regulation of Hepatic Gluconeogenesis by Nuclear Receptor Coactivator 6

doi: 10.14348/molcells.2022.2222

Figure Lengend Snippet: (A) Interactions between CREB and NCOA6. 293T cells were transfected with HA-CREB and FLAG-NCOA6 and lysates were immunoprecipitated from these cells using anti-HA or anti-FLAG antibody. The coimmunoprecipitates were then analyzed by western blotting with anti-HA or anti-FLAG antibody. (B) Sequence alignment of the CRE-containing promoter regions of the Pck1 or G6pc gene among human, mouse, and rat. (C) Schematic diagram of the r Pck1 or h G6PC promoter-Luc reporter containing WT or MT CRE. The red bars indicate the loci of the ChIP primers used in and . (D and E) Effects of NCOA6 on the CREB-mediated transcriptional activation of the r Pck1 (D) and h G6PC (E) promoter (n = 5 independent experiments). WT or MT CRE-containing Pck1 or G6PC promoter-Luc reporters were cotransfected into HepG2 cells with or without HA-CREB, FLAG-NCOA6 and actin-β-galactosidase. Luciferase activities were then measured with a luminometer and normalized using β-galactosidase activities. Data are presented as the mean ± SEM; *** P < 0.001, determined using one-way ANOVA. IP, immunoprecipitation; WT, wild type; MT, mutant; Fsk, forskolin.

Article Snippet: After membrane blocking with 5% skim milk, the membrane was probed with primary antibodies against NCOA6 (Cat. No. NB200-335; Novus Biologicals, USA), PCK1 (Cat. No. sc-32879; Santa Cruz Biotechnology, USA), CREB (Cat. No. 9197; Cell Signaling Technology, USA), p-CREB (Cat. No. 9198S; Cell Signaling Technology), or α-tubulin (Cat. No. T9026; Sigma-Aldrich).

Techniques: Transfection, Immunoprecipitation, Western Blot, Sequencing, Activation Assay, Luciferase, Mutagenesis

Journal: Molecules and Cells

Article Title: Regulation of Hepatic Gluconeogenesis by Nuclear Receptor Coactivator 6

doi: 10.14348/molcells.2022.2222

Figure Lengend Snippet: (A and B) 293T cells were cotransfected with a Pck1 (A) or G6PC (B) promoter-Luc reporter, HA-CREB and FLAG-NCOA6. Modified ChIP assays were then performed using anti-HA, anti-FLAG or IgG antibodies, followed by analysis using conventional PCR (n = 4 independent experiments). The intensities of the PCR bands were measured using the ImageJ program. Data are presented as the mean ± SEM; * P < 0.05, ** P < 0.01, determined by t -test. WT, wild type; MT, mutant; IP, immunoprecipitation. (C) Hypothetical model for the gluconeogenic transcription of the Pck1 and G6pc genes mediated via NCOA6.

Article Snippet: After membrane blocking with 5% skim milk, the membrane was probed with primary antibodies against NCOA6 (Cat. No. NB200-335; Novus Biologicals, USA), PCK1 (Cat. No. sc-32879; Santa Cruz Biotechnology, USA), CREB (Cat. No. 9197; Cell Signaling Technology, USA), p-CREB (Cat. No. 9198S; Cell Signaling Technology), or α-tubulin (Cat. No. T9026; Sigma-Aldrich).

Techniques: Modification, Mutagenesis, Immunoprecipitation

Journal: Frontiers in cell and developmental biology

Article Title: Autophagy-Based Unconventional Secretory for AIM2 Inflammasome Drives DNA Damage Resistance During Intervertebral Disc Degeneration.

doi: 10.3389/fcell.2021.672847

Figure Lengend Snippet: FIGURE 2 | Silencing of AIM2 ameliorates DNA damage in NP cells in vitro. (A) Protein levels and quantification of AIM2, ASC, pro-caspase-1, and cleaved caspase-1 in H2O2-treated (200 µM, 8 h) NP cells. NP cells in the control group were treated with equivalent solvent. (B) Extracellular level of IL-1β and IL-18 measured by ELISA. (C,D) TUNEL analysis of H2O2-treated NP cells (C) and corresponding quantification of cell apoptosis (D). (E) Immunostaining of γ-H2AX in H2O2-treated NP cells. Data were presented as the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group. (F) Protein levels and quantification of AIM2, ASC, pro-caspase-1, and cleaved caspase-1 in H2O2-treated NP cells co-treated with si-AIM2 or si-scr. (G) Extracellular level of IL-1β and IL-18 measured by ELISA. (H,I) Represent images of TUNEL analysis (H) and evaluation of cell apoptotic rates (I). (J) Immunostaining of γ-H2AX in H2O2-treated NP cells co-treated with si-AIM2 or si-scr. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control-si-scr group, n = 3.

Article Snippet: The membranes were blocked with 5% milk for 1 h. The primary antibodies used were as follows: AIM2 (Proteintech, 1:2,000), ASC (Proteintech, 1:2,000), Caspase-1 (CST, 1:1,000), LC3 (Abcam, 1:1,000), GRASP55 (Proteintech, 1:1,000), GRASP65 (Proteintech, 1:1,000), CD63 (Proteintech, 1:500), TSG101 (Proteintech, 1:1,000), and Calnexin (Proteintech, 1:5,000).

Techniques: In Vitro, Control, Solvent, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Immunostaining

Journal: Frontiers in cell and developmental biology

Article Title: Autophagy-Based Unconventional Secretory for AIM2 Inflammasome Drives DNA Damage Resistance During Intervertebral Disc Degeneration.

doi: 10.3389/fcell.2021.672847

Figure Lengend Snippet: FIGURE 8 | Schematic model illustrates the autophagic secretory pathway of AIM2 in NP cells. Oxidative stress-induced DNA damage activates the AIM2 inflammasome and elicits the downstream inflammation cascade. During this process, autophagy activation ameliorates DNA damage via an unconventional secretion of AIM2. AIM2, Absent in melanoma 2; ASC, Apoptosis-associated speck-like protein containing a CARD; NP, Nucleus pulposus; ROS, Reactive oxygen species; IL-1β, Interleukin-1b; IL-18, Interleukin 18; GRASP55, Golgi re-assembly and stacking protein 55.

Article Snippet: The membranes were blocked with 5% milk for 1 h. The primary antibodies used were as follows: AIM2 (Proteintech, 1:2,000), ASC (Proteintech, 1:2,000), Caspase-1 (CST, 1:1,000), LC3 (Abcam, 1:1,000), GRASP55 (Proteintech, 1:1,000), GRASP65 (Proteintech, 1:1,000), CD63 (Proteintech, 1:500), TSG101 (Proteintech, 1:1,000), and Calnexin (Proteintech, 1:5,000).

Techniques: Activation Assay

Journal: Molecular pharmacology

Article Title: THE COACTIVATOR NCOA6 MEDIATES THE SYNERGISTIC ACTIVATION OF HUMAN CYTOCHROME P-450 2C9 BY THE CONSTITUTIVE ANDROSTANE RECEPTOR AND HEPATIC NUCLEAR FACTOR-4?

doi: 10.1124/mol.108.048983

Figure Lengend Snippet: (a) Isolation of HNF4α binding complex from nuclear extracts of HepG2 cells in which Ad-CAR was over expressed (AdCAR-NEs). SDS/gradient gel analysis (4–20%) and silver staining of HNF4α binding complexes from 7.5 mg of AdCAR-NEs bound to GST (lane 2) and GST-HNF4α (lane 4). GST-HNF4α without the addition of NE is shown as a control (lane 3) and 50µg of AdCAR-NE is shown as input (lane 1). (b) Immunoblots identifying CAR and various coactivators in the GST-HNF4α bound complex. Samples from Input: AdCAR expressing NEs, GST: GST bound proteins from AdCAR-NEs and GST-HNF4α bound proteins from AdCAR-NEs were immunoblotted with specific antibodies for CBP, PGC-1α, NCOA6, PIMT and CAR. Lane 1: NE input, lane 2: NE bound to GST as a control and lane 3: GST-HNF4α complex from Ad-CAR-NEs.

Article Snippet: Virus particles were purified on continuous cesium chloride, dialyzed and stored in Tris buffered sucrose. siRNA targets for NCOA6 were identified using Genscript’s target finder, construct builder and sequence scrambler for construction of negative control siRNA.

Techniques: Isolation, Binding Assay, Silver Staining, Control, Western Blot, Expressing

Journal: Molecular pharmacology

Article Title: THE COACTIVATOR NCOA6 MEDIATES THE SYNERGISTIC ACTIVATION OF HUMAN CYTOCHROME P-450 2C9 BY THE CONSTITUTIVE ANDROSTANE RECEPTOR AND HEPATIC NUCLEAR FACTOR-4?

doi: 10.1124/mol.108.048983

Figure Lengend Snippet: The first LXXLL motif of NCOA6 interacts with HNF4α and CAR. (a) GST-CAR and HNF4α were immobilized on GSH-sepharose beads. NCOA6 and PBP as a positive control were translated in vitro in the presence of 35S methionine using Promega’s in vitro system. Full length radiolabeled proteins were allowed to interact with GST, GST-CAR, and GST-HNF4α (+ 100 nM CITCO or DMSO as the control). Bound proteins were heat denatured, separated by electrophoresis on a 4–20% gradient gels, fixed, signal was amplified and dried for autoradiography. 1/10th of the transcription/translation mixes were loaded as input. (b) Schematic representation of NCOA6 domains. GST-NCOA6 domains or GST control were incubated with (c) 35S methionine labeled-CAR (in the presence and absence of 100 nM CITCO) or (d) 35S methionine labeled-HNF4α (no ligand) and subjected to SDS-gel electrophoresis, gels were dried and autoradiographed as described in Methods.

Article Snippet: Virus particles were purified on continuous cesium chloride, dialyzed and stored in Tris buffered sucrose. siRNA targets for NCOA6 were identified using Genscript’s target finder, construct builder and sequence scrambler for construction of negative control siRNA.

Techniques: Positive Control, In Vitro, Control, Electrophoresis, Amplification, Autoradiography, Incubation, Labeling, SDS-Gel

Journal: Molecular pharmacology

Article Title: THE COACTIVATOR NCOA6 MEDIATES THE SYNERGISTIC ACTIVATION OF HUMAN CYTOCHROME P-450 2C9 BY THE CONSTITUTIVE ANDROSTANE RECEPTOR AND HEPATIC NUCLEAR FACTOR-4?

doi: 10.1124/mol.108.048983

Figure Lengend Snippet: (a) NCOA6 does not effect CAR mediated transactivation. HepG2 cells were transfected with CAR, NCOA6 and PGC-1α along with CYP2C9 reporter construct, in the presence (hatched bars) and absence (solid bars) of 100nM CITCO as the ligand with pRL as the internal control. All transfections were performed in triplicate. Values are means of triplicates + SE. CAR significantly upregulates the CYP2C9 promoter in the absence and presence of ligand at *, p<0.05, **,p<0.01, ***, p<0.001. Transfection with PGC-1α and CAR significantly transactivates the CYP2C9 promoter more than CAR alone at †††, p<0.001, while NCOA6 had no effect.

Article Snippet: Virus particles were purified on continuous cesium chloride, dialyzed and stored in Tris buffered sucrose. siRNA targets for NCOA6 were identified using Genscript’s target finder, construct builder and sequence scrambler for construction of negative control siRNA.

Techniques: Transfection, Construct, Control

Journal: Molecular pharmacology

Article Title: THE COACTIVATOR NCOA6 MEDIATES THE SYNERGISTIC ACTIVATION OF HUMAN CYTOCHROME P-450 2C9 BY THE CONSTITUTIVE ANDROSTANE RECEPTOR AND HEPATIC NUCLEAR FACTOR-4?

doi: 10.1124/mol.108.048983

Figure Lengend Snippet: (a) Screening of siRNAs for silencing NCOA6 mRNA in HepG2 cells. Five potential targets for silencing NCOA6 were identified in silico. HepG2 cells were infected with 2.5×109 VP/ml for each of the siRNAs for NCOA6 (NC-I to NC-V) individually with scrambled siRNA as control. After 48h, total RNA was prepared to estimate NCOA6 mRNA levels using qPCR. One target, NC-V was not effective (not shown). All the remaining targets significantly suppressed NCOA6 mRNA, ***, p<0.001, although NC-III was the most effective p<0.001, ††. Values represent means ± SE of triplicate analyses.

Article Snippet: Virus particles were purified on continuous cesium chloride, dialyzed and stored in Tris buffered sucrose. siRNA targets for NCOA6 were identified using Genscript’s target finder, construct builder and sequence scrambler for construction of negative control siRNA.

Techniques: In Silico, Infection, Control

Journal: Molecular pharmacology

Article Title: THE COACTIVATOR NCOA6 MEDIATES THE SYNERGISTIC ACTIVATION OF HUMAN CYTOCHROME P-450 2C9 BY THE CONSTITUTIVE ANDROSTANE RECEPTOR AND HEPATIC NUCLEAR FACTOR-4?

doi: 10.1124/mol.108.048983

Figure Lengend Snippet: (a) SiNCOA6 decreases the endogenous CYP2C9 expression. HepG2 cell were infected with adenovirus containing scrambled or siNCOA6(NC-III) for 48h. The cells were harvested, and total RNA was prepared to estimate constitutive CYP2C9 mRNA levels using qPCR. NC-III significantly decreases endogenous CYP2C9 mRNA at *(p<0.001). (b) Silencing of NCOA6 abolishes the synergistic effects of adenoviral expression of CAR and HNF4α on CYP2C9 mRNA in HepG2 cells. HepG2 cells were infected with adenovirus expressing lacZ, CAR, HNF4α, CAR-HNF4α and CAR-HNF4α with siNCOA6(NC-III) for 48h. The cells were harvested and total RNA was used to measure CYP2C9 mRNA by qPCR. CAR or HNF4α significantly increased CYP2C9 mRNA at *, p<0.05, **p<0.01, ***p<0.001. †††, synergistic rather than additive response to transfection with HNF4α and CAR at p<0.001. ##, NCIII significantly reduced the CYP2C9 mRNA response to HNF4α and CAR. Values represent means ± SE of triplicates.

Article Snippet: Virus particles were purified on continuous cesium chloride, dialyzed and stored in Tris buffered sucrose. siRNA targets for NCOA6 were identified using Genscript’s target finder, construct builder and sequence scrambler for construction of negative control siRNA.

Techniques: Expressing, Infection, Transfection

Journal: Molecular pharmacology

Article Title: THE COACTIVATOR NCOA6 MEDIATES THE SYNERGISTIC ACTIVATION OF HUMAN CYTOCHROME P-450 2C9 BY THE CONSTITUTIVE ANDROSTANE RECEPTOR AND HEPATIC NUCLEAR FACTOR-4?

doi: 10.1124/mol.108.048983

Figure Lengend Snippet: ChIP analysis for CAR-HNF4α mediated recruitment of nuclear receptor cofactors to the CYP2C9 gene promoter. Chromatin extracts isolated from HepG2 cells infected individually with adenovirus expressing, lacZ, CAR, HNF4α, CAR-HNF4α and CAR-HNF4α with siNCOA6 (NC-III) were pre-cleared as described in Material and Methods and immunoprecipitated with antibodies for CAR, HNF4α, NCOA6, PIMT, CBP and PGC-1α . PCR was used to analyze the CYP2C9 promoter at the CAR binding sites (a) and HNF4α binding sites (b) control negative primers (c). Expression of CAR and HNF4α increased their recruitment to their respective sites. Immunoprecipitation with NCOA6, PIMT, CBP and PGC-1α showed their association with both CAR and HNF4α binding sites on CYP2C9 promoter. Silencing of NCOA6 essentially abolishes the recruitment of NCOA6, PIMT, CBP and PGC-1α to the HNF4α sites as well as many of the cofactors to the CAR and HNF4α binding sites.

Article Snippet: Virus particles were purified on continuous cesium chloride, dialyzed and stored in Tris buffered sucrose. siRNA targets for NCOA6 were identified using Genscript’s target finder, construct builder and sequence scrambler for construction of negative control siRNA.

Techniques: Isolation, Infection, Expressing, Immunoprecipitation, Binding Assay, Control

Journal: International Journal of Biological Sciences

Article Title: Identification and Characterization of the Alternatively Spliced Nuclear Receptor Coactivator-6 Isoforms

doi:

Figure Lengend Snippet: Identification of NCOA6 splicing isoforms. A. A part of the mouse NCOA6 gene structure from exon 8 to exon 14. The boxes represent exons. The arrows indicate the locations and directions of the PCR primers. B. Predicted lengths of PCR product from the indicated PCR primer pairs (left panel) and the RT-PCR products amplified from the NCOA6 mRNA of mouse testis using the indicated PCR primer pairs. The alternative splicing isoforms identified from the upper and lower bands in each lane are labeled below the gel image. C. The DNA sequence of mouse exon 10b and human exon 11b in capital letters and its 5' and 3' intron sequences in small letters at the splicing junctions. Stop codons are italicized. n.t., nucleotide sequences; a.a., amino acid sequences coded by exon 10a in mouse and 11a in human. D. Diagram of the four NCOA6 isoforms derived from alternate splicing of human exons 11a and 11b (left panel). The lengths of coding nucleotides and a.a. residues as well as calculated molecular masses of individual isoforms are summarized in the right panel. GenBank accession numbers: JF707634 - JF707639.

Article Snippet: To express GFP-NCOA6 fusion proteins, NCOA6 cDNA fragments were amplified by PCR and ligated to the 3' GFP coding sequence in the pEGFP-C3 plasmid (BD Biosciences Clontech, Mountain View, CA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Labeling, Sequencing, Derivative Assay

Journal: International Journal of Biological Sciences

Article Title: Identification and Characterization of the Alternatively Spliced Nuclear Receptor Coactivator-6 Isoforms

doi:

Figure Lengend Snippet: Expression of the NCOA6 isoforms in mouse tissues. A. Detection of NCOA6 mRNAs in different mouse organs by RT-PCR. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) RT-PCR products served as an endogenous control. B. Analysis of NCOA6 isoform mRNA levels by RT-PCR in the mouse mammary glands at different developmental stages. Vir, P18, L15 and In3, mammary glands from virgin, pregnant day 18, lactation day 15 and involution day 3 female mice, respectively. C. Western blot analysis of mouse testis tissue extracts using antibodies against NCOA6 peptide regions coded by mouse exons 3-4 and exons 12-14, respectively.

Article Snippet: To express GFP-NCOA6 fusion proteins, NCOA6 cDNA fragments were amplified by PCR and ligated to the 3' GFP coding sequence in the pEGFP-C3 plasmid (BD Biosciences Clontech, Mountain View, CA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Identification and Characterization of the Alternatively Spliced Nuclear Receptor Coactivator-6 Isoforms

doi:

Figure Lengend Snippet: Subcellular locations of NCOA6 isoforms. A. Subcellular localizations of GFP-fused hNCOA6 isoforms in transfected Hela and NCOA6 null MEF cells. Live cell images were taken by GFP fluorescent microscopy (left column) and diachronic in contrast image (DIC) microscopy (central column). The merged images are also shown (right column). B. Subcellular locations of Flag-tagged hNCOA6 isoforms in Hela and NCOA6 null MEF cells. FITC (green) signals indicate the cellular locations of the Flag-tagged hNCOA6 isoforms. DAPI staining indicates the cell nuclei. C. Hela cells and NCOA6 null MEF cells were transfected with GFP, GFP-hNCOA6-e13-15 or GFP-hNCOA6-δ expression plasmid. Cells were stained with mitochondrial tracker (red color) and examined by fluorescent and DIC microscopy. Merged images of GFP and mitochondrial (Mit) staining as well as GFP and DIC are shown.

Article Snippet: To express GFP-NCOA6 fusion proteins, NCOA6 cDNA fragments were amplified by PCR and ligated to the 3' GFP coding sequence in the pEGFP-C3 plasmid (BD Biosciences Clontech, Mountain View, CA).

Techniques: Transfection, Microscopy, Staining, Expressing, Plasmid Preparation

Journal: International Journal of Biological Sciences

Article Title: Identification and Characterization of the Alternatively Spliced Nuclear Receptor Coactivator-6 Isoforms

doi:

Figure Lengend Snippet: Identification of NCOA6 isoform sequences responsible for their subcellular localizations. A. Diagram showing a.a. sequences of hNCOA6-α and -γ coded by specific exons, as well as all other constructed and expressed isoform fragments used in mapping the regions responsible for their subcellular localizations. B. Subcellular localizations of Flag-tagged hNCOA6 isoform fragments (green) in transfected Hela and NCOA6 null MEF cells. Cell nuclei were stained with DAPI. C. Subcellular localizations of GFP-fused hNCOA6 isoform fragments.

Article Snippet: To express GFP-NCOA6 fusion proteins, NCOA6 cDNA fragments were amplified by PCR and ligated to the 3' GFP coding sequence in the pEGFP-C3 plasmid (BD Biosciences Clontech, Mountain View, CA).

Techniques: Construct, Transfection, Staining

Journal: International Journal of Biological Sciences

Article Title: Identification and Characterization of the Alternatively Spliced Nuclear Receptor Coactivator-6 Isoforms

doi:

Figure Lengend Snippet: Distribution of NCOA6 isoform proteins in subcellular extracts. A. Transfected Hela cells expressing the indicated Flag-tagged hNCOA6 isoforms or human exon 11a-coded peptide (11a) were subjected to preparation of cytoplasmic (C) and nuclear (N) fractions, followed by Western blot analysis with antibodies against Flag-tagged isoforms, PARP1 (a nuclear protein control) and Hsp90 (a cytosolic protein control). B. Western blot analysis of another batch of cytosolic and nuclear fractions prepared from Hela cells transfected with more plasmid DNA versus panel A. C. Western blot analysis of cytosolic and nuclear fractions prepared from Hela cells expressing NCOA6-γ and -δ proteins. VDAC served as a mitochondrial protein control. D. Western blot analysis of total lysate (T) and purified mitochondrial (Mi) extract of Hela cells transfected with NCOA6-δ.

Article Snippet: To express GFP-NCOA6 fusion proteins, NCOA6 cDNA fragments were amplified by PCR and ligated to the 3' GFP coding sequence in the pEGFP-C3 plasmid (BD Biosciences Clontech, Mountain View, CA).

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

Journal: International Journal of Biological Sciences

Article Title: Identification and Characterization of the Alternatively Spliced Nuclear Receptor Coactivator-6 Isoforms

doi:

Figure Lengend Snippet: Assessment of the coactivator activities of the NCOA6 isoforms. Hela cells were co-transfected with ERα, ERE-tk-Luc reporter and one of the NCOA6 isoform expression plasmids or a mock plasmid as indicated. A β-galactosidase expression vector was also co-transfected as an internal control. Cells were treated with either 17β-estradiol or its vehicle (ethanol) and subjected to luciferase and β-galactosidase activity assays. The luciferase activity was normalized to the β-galactosidase activity. *, P < 0.05 by unpaired t test.

Article Snippet: To express GFP-NCOA6 fusion proteins, NCOA6 cDNA fragments were amplified by PCR and ligated to the 3' GFP coding sequence in the pEGFP-C3 plasmid (BD Biosciences Clontech, Mountain View, CA).

Techniques: Transfection, Expressing, Plasmid Preparation, Luciferase, Activity Assay