Journal: Endocrinology

Article Title: PROX1 Promotes Secretory Granule Formation in Medullary Thyroid Cancer Cells.

doi: 10.1210/en.2015-1973

Figure Lengend Snippet: Figure 5. Effect of PROX1 on known CHGA transcription-regulatory factors. A, A schematic diagram of the CHGA promoter region and the putative PROX1 binding site. CRE, cAMP-responsive element: ex, exon; RE1, repressor element 1; TSS, transcription start site. ChIP-PCR target indicates the region amplified in the chromatin immunoprecipitation- based PCR assay. B, Semiquantitative RT-PCR analysis of PROX1 and CHGA, REST mRNA expression, and Western blot analysis of REST, CREB, phosphorylated CREB (CREB-P), and -actin expression in PROX1-inducible KTC1 and TTA1 cells in the absence (0 h) or presense (12, 24, and 48 h) of the inducer Dox. Data are shown as means SD. C, ChIP-PCR assay of CREB and REST binding to the CHGA gene. Cell lysates of Dox-treated or untreated FLAG-tagged PROX1-transfected KTC1 cells (KTC1-PROX1) were immunoprecipitated by using anti- CREB or anti-PROX1 antibody or normal rabbit IgG. Coprecipitation of gene fragments of CHGA containing CRE or RE1 sequence (CHGA[481 to 367] or CHGA[272 to 808], respectively) were assayed using PCR. CREB, rabbit anti-CREB antibody; IgG, normal rabbit IgG; IP, immunoprecipitation; REST, rabbit anti-REST antibody. The experiment was conducted in triplicate. Data are shown as means SD.

Article Snippet: Briefly, the chromatin solution was incubated with 1 g or recommended amount of mouse anti-FLAG antibody (Sigma-Aldrich), rabbit anti-CREB (Cell Signaling Technology), rabbit anti-REST antibody (Bethyl Laboratories, Inc), normal mouse IgG (Santa Cruz Biotechnology), or normal rabbit IgG (Santa Cruz Biotechnology), and the antibody/chromatin mixtures were then precipitated with Protein G beads.

Techniques: Binding Assay, Amplification, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Transfection, Immunoprecipitation, Sequencing

Journal: Journal of Oncology

Article Title: Modulation of CXCR4, CXCL12, and Tumor Cell Invasion Potential In Vitro by Phytochemicals

doi: 10.1155/2009/491985

Figure Lengend Snippet: Dose-response curves for CXCR4 and CXCL12 mRNAs after treatment of breast and ovarian cancer cells with 1–10 μ M genistein. (a) CXCR4 and CXCL12 mRNA quantification after treatment of MCF-7 cells with increasing concentrations of genistein for 24 hours. (b) CXCR4 quantification after treatment of MDA-MB-231 cells with genistein for 24 hours. MDA-MB-231 cells do not express CXCL12 as determined by real-time PCR. (c) CXCR4 and CXCL12 quantification after treatment of BG-1 cells with increasing doses of genistein for 24 hours. All data were normalized to corresponding mRNA quantities for the 36B4 housekeeping gene. * P < .05, ** P < .01, *** P < .001; relative to 0 μ M genistein control.

Article Snippet: CXCR4 and CXCL12 cDNAs were amplified using Assays on Demand (Affinity Bioreagents, Golden, Colo, USA; product numbers Hs00607978_s1 and Hs00171022_m1, resp. ; sequences proprietary).

Techniques: Real-time Polymerase Chain Reaction, Control

Journal: Journal of Oncology

Article Title: Modulation of CXCR4, CXCL12, and Tumor Cell Invasion Potential In Vitro by Phytochemicals

doi: 10.1155/2009/491985

Figure Lengend Snippet: Genistein downregulates CXCR4 surface expression and intracellular CXCL12 expression. Downregulation of CXCR4 protein expression in MCF-7, MDA-MB-231, and BG-1 cells at concentrations of genistein ranging from 0–100 μ M for a 24-hour period (a)–(c). Downregulation of intracellular CXCL12 protein expression in (a) MCF-7 and (c) BG-1 cells at 30–100 μ M genistein. Fluorescence distribution plots depict one representative experiment. Isotype controls are represented by filled histograms. Mean fluorescence intensities (MFI) and their corresponding standard deviations are derived from three independent experiments.

Article Snippet: CXCR4 and CXCL12 cDNAs were amplified using Assays on Demand (Affinity Bioreagents, Golden, Colo, USA; product numbers Hs00607978_s1 and Hs00171022_m1, resp. ; sequences proprietary).

Techniques: Expressing, Fluorescence, Derivative Assay

Journal: Journal of Oncology

Article Title: Modulation of CXCR4, CXCL12, and Tumor Cell Invasion Potential In Vitro by Phytochemicals

doi: 10.1155/2009/491985

Figure Lengend Snippet: In combination, genistein and DIM are more efficacious in the downregulation of CXCR4 and CXCL12 mRNAs than either compound alone. The effect of downregulation of CXCR4 in (a) MDA-MB-231 and (b) BG-1 cells by 20 μ M DIM and 100 μ M genistein in combination is greater than their individual effects. The same is seen in (b) BG-1 cells after CXCL12 quantification. Ctl = DMSO alone; ** P < .01, *** P < .001.

Article Snippet: CXCR4 and CXCL12 cDNAs were amplified using Assays on Demand (Affinity Bioreagents, Golden, Colo, USA; product numbers Hs00607978_s1 and Hs00171022_m1, resp. ; sequences proprietary).

Techniques:

Journal: Frontiers in Microbiology

Article Title: PCR performance of a thermostable heterodimeric archaeal DNA polymerase

doi: 10.3389/fmicb.2014.00195

Figure Lengend Snippet: Properties of experimental thermophilic DNA polymerases .

Article Snippet: Pab-polB ( Isis DNA polymerase) and Taq-polA ( Taq DNA polymerase) were purchased from MP biomedicals.

Techniques:

Journal: Frontiers in Microbiology

Article Title: PCR performance of a thermostable heterodimeric archaeal DNA polymerase

doi: 10.3389/fmicb.2014.00195

Figure Lengend Snippet: Effect of input genomic DNA on PCR efficiency and specificity . PCR amplification of the 0.5 kb target (Table ) was carried out with 0.1 U of Pab-polD (A) , 1 U of Pab-polB (B) , and 1 U of Taq-polA (C) in their respective reaction buffer (Table ). PCR program was (2 min at 94°C) × 1; (1 min at 94°C, 1 min at 58°C, 2 min at 72°C) × 30; (5 min at 72°C) × 1. Molecular weight markers (M) are SmartLadder SF from Eurogentec. The arrow indicates the specific 0.5 kb band.

Article Snippet: Pab-polB ( Isis DNA polymerase) and Taq-polA ( Taq DNA polymerase) were purchased from MP biomedicals.

Techniques: Amplification, Molecular Weight

Journal: Frontiers in Microbiology

Article Title: PCR performance of a thermostable heterodimeric archaeal DNA polymerase

doi: 10.3389/fmicb.2014.00195

Figure Lengend Snippet: Impact of thermal denaturation during cycling . PCR amplification of the 0.5 kb target (Table ) was carried out with 0.1 U of Pab-polD (A) , 1 U of Pab-polB (B) , and 1 U of Taq-polA (C) in their respective reaction buffer (Table ). PCR program was (2 min at 94°C) × 1; (1 min at the indicated temperature, 1 min at 58°C, 2 min at 72°C) × 30; (5 min at 72°C) × 1. Molecular weight markers (M) are SmartLadder SF from Eurogentec.

Article Snippet: Pab-polB ( Isis DNA polymerase) and Taq-polA ( Taq DNA polymerase) were purchased from MP biomedicals.

Techniques: Amplification, Molecular Weight

Journal: Frontiers in Microbiology

Article Title: PCR performance of a thermostable heterodimeric archaeal DNA polymerase

doi: 10.3389/fmicb.2014.00195

Figure Lengend Snippet: Rate of DNA extension . PCR amplification of the 1.95 kb target (Table ) was carried out with 0.1 U of Pab-polD (A) , 1 U of Pab-polB (B) , and 1 U of Taq-polA (C) in their respective reaction buffer (Table ). PCR program was (2 min at 94°C) × 1; (1 min at 94°C, 1 min at 58°C, varying times in seconds as indicated at 72°C) × 30. Molecular weight markers (M) are SmartLadder LF from Eurogentec. The arrow indicates the specific 1.95 kb band.

Article Snippet: Pab-polB ( Isis DNA polymerase) and Taq-polA ( Taq DNA polymerase) were purchased from MP biomedicals.

Techniques: Amplification, Molecular Weight

Journal: Frontiers in Microbiology

Article Title: PCR performance of a thermostable heterodimeric archaeal DNA polymerase

doi: 10.3389/fmicb.2014.00195

Figure Lengend Snippet: PCR amplification of DNA fragments of various lengths . PCR reactions were carried out using 100 ng of genomic DNA with 0.1 Uof Pab-polD (A) , 1 U of Pab-polB (B) , and 1 U of Taq-polA (C) in their respective reaction buffer (Table ). Primer sets were chosen to amplify 0.5, 1.1, 1.95, 2.95, 4.15, and 10 kb (Table ). PCR programs were (2 min at 94°C) × 1; (1 min at 94°C/1 min at 58°C/2, 4, 6, 8, 11, and 16 min with respect to the target length at 72°C) × 30. Molecular weight markers (M) are SmartLadder LF from Eurogentec.

Article Snippet: Pab-polB ( Isis DNA polymerase) and Taq-polA ( Taq DNA polymerase) were purchased from MP biomedicals.

Techniques: Amplification, Molecular Weight

Journal: Frontiers in Microbiology

Article Title: PCR performance of a thermostable heterodimeric archaeal DNA polymerase

doi: 10.3389/fmicb.2014.00195

Figure Lengend Snippet: PCR amplification in the presence of primer mismatches . PCR amplification of the 0.5 kb target (Table ) was carried out with 0.1 U of Pab-polD (A) , 1 U of Pab-polB (B) , and 1 U of Taq-polA (C) in their respective reaction buffer (Table ). Primer sets were chosen to introduce 0, 1, 2, 3, and 4 mismatches at the 3'-termini of the forward primer (Table ). PCR program was (2 min at 94°C) × 1; (1 min at 94°C, 1 min at 58°C, 2 min at 72°C) × 30; (5 min at 72°C) × 1. The arrow indicates the specific 0.5 kb band.

Article Snippet: Pab-polB ( Isis DNA polymerase) and Taq-polA ( Taq DNA polymerase) were purchased from MP biomedicals.

Techniques: Amplification, Introduce

Journal: Frontiers in Microbiology

Article Title: PCR performance of a thermostable heterodimeric archaeal DNA polymerase

doi: 10.3389/fmicb.2014.00195

Figure Lengend Snippet: Potential inhibitory effects of organic and inorganic substances on PCR .

Article Snippet: Pab-polB ( Isis DNA polymerase) and Taq-polA ( Taq DNA polymerase) were purchased from MP biomedicals.

Techniques: Concentration Assay

Journal: Cell Death Discovery

Article Title: Cigarette smoke activates the parthanatos pathway of cell death in human bronchial epithelial cells

doi: 10.1038/s41420-019-0205-3

Figure Lengend Snippet: PARP–1 recognition of smoke-mediated DNA strand breaks activates itself to poly-adenosylate (PAR) itself and other nuclear proteins . Extensive DNA damage resulting in PARP–1 overactivation produces branched chains of PAR . Poly (ADP-ribose) glycohydrolase (PARG) releases PAR by cleavage that unless hydrolyzed by ADP-ribosylhydrolase 3 (ARH3) induces mitochondrial proteins Apoptosis-Inducing Factor (AIF) and EndonucleaseG (EndoG) to translocate to the nucleus causing genomic DNA fragmentation

Article Snippet: Samples were amplified on a CFX-96 Bio-Rad real time PCR machine using SsoAdvanced universal probes supermix (172–5284, Bio-Rad) and TaqMan primers for PARP-1 and GAPDH as housekeeping gene (Hs00242302_m1 resp. Hs02758991_g1, Thermo Fisher Scientific).

Techniques:

Journal: Cell Death Discovery

Article Title: Cigarette smoke activates the parthanatos pathway of cell death in human bronchial epithelial cells

doi: 10.1038/s41420-019-0205-3

Figure Lengend Snippet: a RT-PCR of PARP-1 normalized to GAPDH in HBE cells from non-smokers ( n = 12) and smokers ( n = 17). b Western blot analysis of PARP-1 protein expression normalized to β-actin in HBE cells from non-smokers ( n = 6) and smokers ( n = 12). Plotted individual data points with line representing mean

Article Snippet: Samples were amplified on a CFX-96 Bio-Rad real time PCR machine using SsoAdvanced universal probes supermix (172–5284, Bio-Rad) and TaqMan primers for PARP-1 and GAPDH as housekeeping gene (Hs00242302_m1 resp. Hs02758991_g1, Thermo Fisher Scientific).

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

Journal: The Journal of Biological Chemistry

Article Title: REST Regulates DYRK1A Transcription in a Negative Feedback Loop *

doi: 10.1074/jbc.M110.174540

Figure Lengend Snippet: REST activates human DYRK1A gene transcription. A, pDYluc-A contains the functional promoter of DYRK1A gene. The promoter construct pDYluc-A, containing the 1127-bp fragment from the human DYRK1A gene 5′-UTR, was transfected into HEK293 cells. Luciferase activity was measured 24 h after transfection by a luminometer. The values represent the means ± S.E. (n = 3). *, p < 0.001 by Student's t test. B, DYRK1A gene promoter is activated by REST. REST was co-transfected with pDYluc-A into HEK293 cells. Luciferase assay was performed 24 h after transfection. The values represent the means ± S.E. (n = 3). *, p < 0.001 by Student's t test. C, RT-PCR showed that REST knockdown construct psiREST can knock down Rest mRNA expression. β-Actin was used as the internal control. The values represent the means ± S.E. (n = 3). *, p = 0.0006 by Student's t test. D, the knockdown effect of psiREST was verified in protein level by Western blot. HEK293 cells were co-transfected with REST expression plasmid and psiREST. REST was detected with anti-REST antibody. β-Actin was used as loading control. The values represent the means ± S.E. (n = 3). *, p = 0.04 by Student's t test. E, RT-PCR showed that BDNF mRNA was repressed by REST overexpression in HEK293 cells. β-Actin was amplified as the internal control. *, p < 0.001 by Student's t test. F, RT-PCR showed that REST overexpression significantly increased DYRK1A mRNA, whereas REST knockdown significantly decreased DYRK1A mRNA levels. β-Actin was amplified as the internal control. The values represent the means ± S.E. (n = 3). *, p < 0.001 by Student's t test. G, real time PCR showed that DYRK1A expression was elevated with REST overexpression. TaqMan gene expression assay of DYRK1A was performed with an ABI 7900HT real time PCR system. 18 S rRNA was chosen for the internal control. The values represent the means ± S.E. (n = 4). *, p = 0.0032 by Student's t test. Con, control.

Article Snippet: The slices were immunostained with rabbit anti-DYRK1A (1:25; Cell Signaling Technology, Danvers, MA), rabbit anti-REST (1:500; Upstate), or mouse anti-MAP-2 (1:100; Vector Laboratories, Burlingame, CA) primary antibodies at 4 °C overnight.

Techniques: Functional Assay, Construct, Transfection, Luciferase, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Plasmid Preparation, Over Expression, Amplification, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: REST Regulates DYRK1A Transcription in a Negative Feedback Loop *

doi: 10.1074/jbc.M110.174540

Figure Lengend Snippet: Identification of a functional NRSE site in human DYRK1A promoter. A, schematic diagram of the human DYRK1A promoter deletion constructs consisting of a 5′-flanking region with serial deletions cloned into pGL3-Basic in front of the luciferase gene (Luc). Arrowheads indicate the directions of transcription. The numbers represent the end points of each construct. The positions of three putative NRSE sites are shown at the bottom. B, the deletion plasmids were confirmed by sequencing and restriction enzyme digestion checking on a 1.5% agarose gel. Vector size is 4.7 kb, and the DYRK1A gene 5′-flanking fragment inserts range from 0.5 to 1.5 kb. C, the deletion plasmids were transfected into REST-competent C6 or REST-incompetent PC12 cells. Plasmid pRL-TK was used to normalize the transfection efficiency, and luciferase activity was measured at 24 h by a luminometer. The values represent the means ± S.E. (n = 5). *, p < 0.001 by Student's t test. D, PC12 cells were co-transfected with REST expression vector and various DYRK1A promoter deletion constructs. Luciferase assay was performed 24 h after transfection. The values represent the means ± S.E. (n = 5). *, p < 0.001 by Student's t test. E, pDYlucm was made to contain the mutant NRES site of DYRK1A promoter. The mutant plasmid, as well as the wild type pDYluc-A, was co-transfected with REST expression plasmid into HEK293 cells. Plasmid pRL-TK was used to normalize the transfection efficiency, and luciferase activity was measured at 24 h by a luminometer. The values represent the means ± S.E. (n = 3). *, p < 0.01 by Student's t test. F and G, EMSA was performed with IRDye 700-labeled consensus NRSE oligonucleotides (F) or NRSE site of DYRK1A promoter (G). First lane, labeled probe without nuclear extract. Second lane, incubation of infrared labeled NRSE probe with HeLa nuclear extracts retarded the migration rate of the labeled probe, which formed a new shifted DNA-protein complex band. The addition of anti-REST antibody further shifted the band to a larger mass shown by a longer running time (third lane in the lower panel of G). Competition assays were performed by further adding different concentrations of molar excess of unlabeled competitive oligonucleotides. H, anti-REST antibody was used to immunoprecipitate the cross-linked REST-DNA complex in ChIP assay in SH-SY5Y cells. A pair of primers targeting DYRK1A was used to amplify NRSE-DY1 and NRSE-DY3. Signals amplified from input were used as size markers. Mouse IgG was used as a negative control. Con, control.

Article Snippet: The slices were immunostained with rabbit anti-DYRK1A (1:25; Cell Signaling Technology, Danvers, MA), rabbit anti-REST (1:500; Upstate), or mouse anti-MAP-2 (1:100; Vector Laboratories, Burlingame, CA) primary antibodies at 4 °C overnight.

Techniques: Functional Assay, Construct, Clone Assay, Luciferase, Sequencing, Agarose Gel Electrophoresis, Plasmid Preparation, Transfection, Activity Assay, Expressing, Mutagenesis, Labeling, Incubation, Migration, Amplification, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: REST Regulates DYRK1A Transcription in a Negative Feedback Loop *

doi: 10.1074/jbc.M110.174540

Figure Lengend Snippet: DYRK1A and REST coordinately expressed in mouse brain during neurodevelopment and in cell lines. A and B, immunofluorescence staining was performed on coronal slices from adult mouse brain. A Cy3-conjugated anti-rabbit secondary antibody was used to visualize DYRK1A (A1 and B1, red) and REST (A4 and B5, red). MAP2 (microtubule-associated protein 2) was stained to serve as a neuronal marker. A fluorescein-conjugated anti-mouse secondary antibody was used to visualize MAP2 (B2 and B6, green). The nuclei were counterstained with blue DAPI (A2, A5, B3, and B7). Overlay showed that both DYRK1A and REST are enriched in neurons in hippocampus (A3 and A6) and cerebral cortex (B4 and B8). The images were captured with a Leica fluorescence microscope at ×400 (A) or ×600 (B). C, quantitative RT-PCR was performed on cDNA templates prepared from normal mouse brain aging at embryonic days 13 (E13) and 17 (E17); at P1, P7, P14, and P30; and in adult mice. One to three mice were used in each time point as indicated by the numbers after the hyphens. p = 0.0028 by correlation test. Rest (D) and Dyrk1a (E) mRNA levels were determined by RT-PCR in C6, N2A, and PC12 cells. β-Actin was amplified as the internal control. F, quantification of D and E showed that Rest and Dyrk1a were coordinately expressed at low levels in PC12 cells and at high levels in N2a and C6 cells. The values represent the means ± S.E. (n = 3). *, p = 0.0002 by correlation test.

Article Snippet: The slices were immunostained with rabbit anti-DYRK1A (1:25; Cell Signaling Technology, Danvers, MA), rabbit anti-REST (1:500; Upstate), or mouse anti-MAP-2 (1:100; Vector Laboratories, Burlingame, CA) primary antibodies at 4 °C overnight.

Techniques: Immunofluorescence, Staining, Marker, Fluorescence, Microscopy, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Amplification

Journal: The Journal of Biological Chemistry

Article Title: REST Regulates DYRK1A Transcription in a Negative Feedback Loop *

doi: 10.1074/jbc.M110.174540

Figure Lengend Snippet: REST expression is down-regulated by DYRK1A protein imbalance. A, psi-DY (siRNA against DYRK1A) was co-transfected with DYRK1A expression plasmid into N2a cells. DYRK1A was detected by anti-FLAG (M2) antibody. β-Actin was used as loading control. The values represent the means ± S.E. (n = 3). *, p < 0.0001 by Student's t test. B, N2a cells were transfected with DYRK1A expression vector pDYRK1A or knockdown vector psi-DY. Rest mRNA expression was detected by RT-PCR. β-Actin was amplified as the internal control. The values represent the means ± S.E. (n = 3). *, p < 0.0001 by Student's t test. C, N2a cells were co-transfected with REST expression vector and pDYRK1A or psi-DY. The cell lysates were separated with 12% SDS-PAGE. REST expression was detected by anti-REST antibody. β-Actin was used as loading control. The values represent the means ± S.E. (n = 3). *, p < 0.01 by Student's t test. D, HEK293 cells co-transfected with REST expression vector, pUbi-His, and pDYRK1A or psi-DY were chased with 50 μg/ml cycloheximide (CHX) for 1 and 2 h. REST expression was detected by anti-REST antibody. β-Actin was used as loading control. E, HEK293 cells co-transfected with REST expression vector and pDYRK1A or psi-DY were lysed and immunoprecipitated with anti-ubiquitin antibody (1:1000) and detected with anti-REST antibody. The values represent the means ± S.E. (n = 3). *, p < 0.01 by Student's t test. F, N2a cells were co-transfected with C terminus REST truncated mutant, FLAG-tagged REST-FS, together with pDYRK1A or psi-DY. REST-FS protein was detected by anti-FLAG (M2) antibody. β-Actin was used as loading control. The values represent the means ± S.E. (n = 3). p > 0.05 by Student's t test. Con, control; IP, immunoprecipitation.

Article Snippet: The slices were immunostained with rabbit anti-DYRK1A (1:25; Cell Signaling Technology, Danvers, MA), rabbit anti-REST (1:500; Upstate), or mouse anti-MAP-2 (1:100; Vector Laboratories, Burlingame, CA) primary antibodies at 4 °C overnight.

Techniques: Expressing, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Amplification, SDS Page, Immunoprecipitation, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: REST Regulates DYRK1A Transcription in a Negative Feedback Loop *

doi: 10.1074/jbc.M110.174540

Figure Lengend Snippet: Transcriptional activity of REST was reduced by DYRK1A protein imbalance. A and B, EMSA showed that REST activity was perturbed by DYRK1A imbalance. N2a cells were transfected with pDYRK1A and psi-DY (A) or co-transfected with REST expression vector (B). EMSA was performed with IRDye 700-labeled consensus NRSE. Nuclear extracts were obtained from N2a cells transfected with pDYRK1A (lane 2), psi-DY (lane 3), or empty vector (lane 4). The specificity of REST-NRSE binding was indicated by competition by consensus NRSE oligonucleotides (lane 5) or NRSE mutant oligonucleotides (lane 6). C and D, N2a cells were transfected with DYRK1A expression vector pDYRK1A or knockdown vector psi-DY. Bdnf mRNA levels were detected by RT-PCR. β-Actin was amplified as the internal control. The values represent the means ± S.E. (n = 3). *, p < 0.001 by Student's t test. E, the BDNF promoter construct pBDNFluc was co-transfected with pDYRK1A or psi-DY. Luciferase assay was performed 24 h after transfection. The values represent the means ± S.E. (n = 3). *, p < 0.001 by Student's t test. F, the DYRK1A promoter construct pDYluc-A was co-transfected with pDYRK1A or psi-DY into N2a cells. Luciferase activity was measured at 24 h by a luminometer. The values represent the means ± S.E. (n = 4). *, p < 0.001 by Student's t test. Con, control.

Article Snippet: The slices were immunostained with rabbit anti-DYRK1A (1:25; Cell Signaling Technology, Danvers, MA), rabbit anti-REST (1:500; Upstate), or mouse anti-MAP-2 (1:100; Vector Laboratories, Burlingame, CA) primary antibodies at 4 °C overnight.

Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation, Labeling, Binding Assay, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Amplification, Construct, Luciferase

Journal: The Journal of Biological Chemistry

Article Title: REST Regulates DYRK1A Transcription in a Negative Feedback Loop *

doi: 10.1074/jbc.M110.174540

Figure Lengend Snippet: Schematic diagram of the cross-interaction of REST and DYRK1A. The imbalance of DYRK1A expression in cells increases the phosphorylation and subsequent ubiquitination of REST protein. Ubiquitinated REST can be degraded by proteasome, which results in the weaker transcription of DYRK1A via a NRSE site located at DYRK1A gene promoter. Thus, a negative feedback loop is formed in DYRK1A gene regulation.

Article Snippet: The slices were immunostained with rabbit anti-DYRK1A (1:25; Cell Signaling Technology, Danvers, MA), rabbit anti-REST (1:500; Upstate), or mouse anti-MAP-2 (1:100; Vector Laboratories, Burlingame, CA) primary antibodies at 4 °C overnight.

Techniques: Expressing