p2x 1 reporter plasmid  (Millipore)

Journal: PLoS ONE

Article Title: Sensory Dysfunction of Bladder Mucosa and Bladder Oversensitivity in a Rat Model of Metabolic Syndrome

doi: 10.1371/journal.pone.0045578

Figure Lengend Snippet: Western blot analysis with specific antibodies to the TRPV1 receptor, purinergic P2X 2 receptor, and P2X 3 receptor of the rat mucosal layer and the purinergic P2X 1 receptor of the rat smooth muscle layer in controls and FFRs with different in cystometric presentations. A. TRPV1 receptor: The TRPV1 antibody produced a clear single band at 95kDa. B. Purinergic P2X 2 receptor C. Purinergic P2X 3 mature receptor: the predominant P2X 3 form (65kDa). D. Up-regulation of P2X3 native and intermediate polypeptides (up to 55kD) were shown in both FFR groups. E. Purinergic P2X 1 receptor Experiments were repeated two times and representative blots are shown. Data of proteins expression (ratios of signal intensities of investigated receptors relative to β-actin or GAPDH) were calculated with 8 samples in each group. These data of Mean ± SE were standardized and expressed in percentage in which the value of the control group is treated as 100%. Theses values were shown in the bar graph. An asterisk indicates a significant difference between controls and FFR groups (One-way ANOVA with Dunnett’s test, p<0.05).

Article Snippet: Antibodies raised against TRPV1 receptor (Alomone, Israel; 1∶1000 dilution), P2X 2 receptor (Alomone, Israel; 1∶500 dilution), P2X 3 receptor (Neuromics, MN; 1∶500 dilution), P2X 1 receptor (Alomone, Israel; 1∶500 dilution), iNOS (Cayman Chemical, Michigan; 1∶500 dilution), eNOS (BD Biosciences, CA; 1∶1000 dilution), GAPDH (Millipore, CA; 1∶10000 dilution ) and β-actin (Millipore, CA; 1∶10000 dilution) were used.

Techniques: Western Blot, Produced, Expressing

Journal: iScience

Article Title: Mechanisms of HIV-mediated blood-brain barrier compromise and leukocyte transmigration under the current antiretroviral era

doi: 10.1016/j.isci.2024.109236

Figure Lengend Snippet:

Article Snippet: P2X 1 , ThermoFisher Scientific , Cat# PA577662; RRID: AB_2736289.

Techniques: Isolation, Derivative Assay, Recombinant, Saline, Plasmid Preparation, Lysis, SYBR Green Assay, Membrane, Enzyme-linked Immunosorbent Assay, Software

Journal: Poultry Science

Article Title: The release of zearalenone-induced heterophil extracellular traps in chickens is associated with autophagy, glycolysis, PAD enzyme, and P2X 1 receptor

doi: 10.1016/j.psj.2023.102946

Figure Lengend Snippet: The effect of PAD enzyme and P2X 1 receptor in ZEA-triggered HET formation. Heterophils (2 × 10 5 /well) were pretreated with the inhibitor of PAD (Cl-amidine), P2X 1 (NF449) for 30 min, followed by exposure to ZEA (80 μM) for 2 h. Extracellular DNA was quantified by PicoGreen-derived fluorescence intensities using the fluorescence plate reader at 485 nm excitation/525 nm emission. One-way ANOVA was used to determine the significance ( n = 6, **** P < 0.0001).

Article Snippet: The inhibitor of mTOR (Rapamycin, 50 μM), extracellular regulated protein kinases ( ERK ) signaling (U0126, 50 μM), PAD enzyme (Cl-amidine, 6 μM), P2X 1 receptor (NF449, 10 μM), PI3K class I (wortmannin, 50 μM) were obtained from MedChemExpress (New Jersey).

Techniques: Derivative Assay, Fluorescence

Journal: Frontiers in Immunology

Article Title: Activated P2X receptors can up-regulate the expressions of inflammation-related genes via NF-κB pathway in spotted sea bass ( Lateolabrax maculatus )

doi: 10.3389/fimmu.2023.1181067

Figure Lengend Snippet: Sequence Characterization of Lm P2Xs.

Article Snippet: The pcDNA3.1- Lm P2Xs plasmid or pcDNA3.1 plasmid was co-transfected with the pGL4.32 (luc2P/NF-κB-RE/Hygro, NF-κB reporter plasmid, Promega) and pRL-TK (renilla luciferase control plasmid, Promega) into HEK 293T cells.

Techniques: Sequencing

Journal: Frontiers in Immunology

Article Title: Activated P2X receptors can up-regulate the expressions of inflammation-related genes via NF-κB pathway in spotted sea bass ( Lateolabrax maculatus )

doi: 10.3389/fimmu.2023.1181067

Figure Lengend Snippet: Multiple sequence alignment analysis of P2Xs. The N-terminal region, two transmembrane domains (TM1-2), the extracellular loop (ECL), and the C-terminal region of Lm P2Xs are marked above the alignment. Symbol (▴) indicates the conserved cysteine residues. The PKC site, P2X family signature motif, YXXXK motif, and LPS/lipid-binding domain are boxed in orange, pink, red, and green, respectively.

Article Snippet: The pcDNA3.1- Lm P2Xs plasmid or pcDNA3.1 plasmid was co-transfected with the pGL4.32 (luc2P/NF-κB-RE/Hygro, NF-κB reporter plasmid, Promega) and pRL-TK (renilla luciferase control plasmid, Promega) into HEK 293T cells.

Techniques: Sequencing, Binding Assay

Journal: Frontiers in Immunology

Article Title: Activated P2X receptors can up-regulate the expressions of inflammation-related genes via NF-κB pathway in spotted sea bass ( Lateolabrax maculatus )

doi: 10.3389/fimmu.2023.1181067

Figure Lengend Snippet: Phylogenetic tree analysis of Lm P2Xs with the selected P2X family members. Phylogenetic tree was constructed using the NJ method within the MEGAX program and run for 10,000 replications. The percentages of bootstrap values for branches are indicated and the Lm P2Xs are shown in bold.

Article Snippet: The pcDNA3.1- Lm P2Xs plasmid or pcDNA3.1 plasmid was co-transfected with the pGL4.32 (luc2P/NF-κB-RE/Hygro, NF-κB reporter plasmid, Promega) and pRL-TK (renilla luciferase control plasmid, Promega) into HEK 293T cells.

Techniques: Construct

Journal: Frontiers in Immunology

Article Title: Activated P2X receptors can up-regulate the expressions of inflammation-related genes via NF-κB pathway in spotted sea bass ( Lateolabrax maculatus )

doi: 10.3389/fimmu.2023.1181067

Figure Lengend Snippet: Gene synteny of P2X2 (A) , P2X4/7 (B) , and P2X5 (C) . The Lm P2Xs genome sequence data were obtained from the spotted sea bass genome database ( https://www.ncbi.nlm.nih.gov/-genome/43909 ). Synteny information for other vertebrates was retrieved from the Ensembl database ( http://www.ensembl.org/index.html ). Arrows indicate transcription orientations.

Article Snippet: The pcDNA3.1- Lm P2Xs plasmid or pcDNA3.1 plasmid was co-transfected with the pGL4.32 (luc2P/NF-κB-RE/Hygro, NF-κB reporter plasmid, Promega) and pRL-TK (renilla luciferase control plasmid, Promega) into HEK 293T cells.

Techniques: Sequencing

Journal: Frontiers in Immunology

Article Title: Activated P2X receptors can up-regulate the expressions of inflammation-related genes via NF-κB pathway in spotted sea bass ( Lateolabrax maculatus )

doi: 10.3389/fimmu.2023.1181067

Figure Lengend Snippet: Expression analysis of Lm P2Xs in tissues. The expression levels of each gene were normalized to that of EF1α. Data are presented as mean + SEM (N= 4).

Article Snippet: The pcDNA3.1- Lm P2Xs plasmid or pcDNA3.1 plasmid was co-transfected with the pGL4.32 (luc2P/NF-κB-RE/Hygro, NF-κB reporter plasmid, Promega) and pRL-TK (renilla luciferase control plasmid, Promega) into HEK 293T cells.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Activated P2X receptors can up-regulate the expressions of inflammation-related genes via NF-κB pathway in spotted sea bass ( Lateolabrax maculatus )

doi: 10.3389/fimmu.2023.1181067

Figure Lengend Snippet: Expressions of Lm P2Xs in primary head kidney leucocytes after stimulation with PGN, LPS, poly (I:C) or ATP. Primary head kidney leukocytes were isolated from the spotted sea bass head kidney and stimulated with PGN (50 μg/mL) LPS (100 μg/mL), LPS (100 μg/mL), Poly (I:C) (50 μg/mL), 100 μM ATP, 1 mM ATP, or PBS (control). The total RNA was extracted for qPCR analysis. The relative expression levels of target genes were normalized to that of EF1α and fold change was obtained by comparing the relative gene expression level of the treated group and control group (defined as 1). The data are shown as mean +SEM (N=3). *P<0.05 and **P<0.01 are considered significant difference.

Article Snippet: The pcDNA3.1- Lm P2Xs plasmid or pcDNA3.1 plasmid was co-transfected with the pGL4.32 (luc2P/NF-κB-RE/Hygro, NF-κB reporter plasmid, Promega) and pRL-TK (renilla luciferase control plasmid, Promega) into HEK 293T cells.

Techniques: Isolation, Expressing

Journal: Frontiers in Immunology

Article Title: Activated P2X receptors can up-regulate the expressions of inflammation-related genes via NF-κB pathway in spotted sea bass ( Lateolabrax maculatus )

doi: 10.3389/fimmu.2023.1181067

Figure Lengend Snippet: Effect of activated Lm P2Xs on expressions of pro-inflammatory cytokines (A) and apoptosis-related genes (B) in the HEK 293T cells. HEK 293T cells were transfected with pcDNA3.1- Lm P2Xs or pcDNA3.1. After 24 h, the cells were stimulated with (0.1 mM, 1 mM, or 5 mM) and were collected after 6 h. The empty plasmid transfected cells by PBS stimulation were served as control. The relative expression levels of target genes were normalized to that of βactin and fold change was obtained by comparing the relative gene expression level of the treated group and control group (defined as 1). The data are shown as mean +SEM (N=3). *P<0.05 and **P<0.01 are considered significant difference.

Article Snippet: The pcDNA3.1- Lm P2Xs plasmid or pcDNA3.1 plasmid was co-transfected with the pGL4.32 (luc2P/NF-κB-RE/Hygro, NF-κB reporter plasmid, Promega) and pRL-TK (renilla luciferase control plasmid, Promega) into HEK 293T cells.

Techniques: Transfection, Plasmid Preparation, Expressing

Journal: Frontiers in Immunology

Article Title: Activated P2X receptors can up-regulate the expressions of inflammation-related genes via NF-κB pathway in spotted sea bass ( Lateolabrax maculatus )

doi: 10.3389/fimmu.2023.1181067

Figure Lengend Snippet: Effect of activated Lm P2Xs on activity of NF-κB promoter in HEK 293T cells. The pGL4.32 and pRL-TK (negative control) were co-transfected with pcDNA3.1- Lm P2Xs plasmid or pcDNA3.1 plasmid into HEK 293T cells for 24 h. Then the cells were stimulated with ATP (0.1 mM, 1 mM, or 5 mM) or PBS (control) and collected after 6 h. Fold change was obtained by comparing the relative Luciferase activity level of the stimulated group and control group (defined as 1). The data are shown as mean +SEM (N=3). *P<0.05 is considered significant difference.

Article Snippet: The pcDNA3.1- Lm P2Xs plasmid or pcDNA3.1 plasmid was co-transfected with the pGL4.32 (luc2P/NF-κB-RE/Hygro, NF-κB reporter plasmid, Promega) and pRL-TK (renilla luciferase control plasmid, Promega) into HEK 293T cells.

Techniques: Activity Assay, Negative Control, Transfection, Plasmid Preparation, Luciferase

Journal: Frontiers in Immunology

Article Title: Activated P2X receptors can up-regulate the expressions of inflammation-related genes via NF-κB pathway in spotted sea bass ( Lateolabrax maculatus )

doi: 10.3389/fimmu.2023.1181067

Figure Lengend Snippet: Sequence Characterization of Lm P2Xs.

Article Snippet: HEK 293T cells were then transfected with pcDNA3.1- Lm P2Xs or pcDNA3.1 using the PEI 40K Transfection Reagent (Servicebio, China) according to the manufacturer’s instruction.

Techniques: Sequencing

Journal: Frontiers in Immunology

Article Title: Activated P2X receptors can up-regulate the expressions of inflammation-related genes via NF-κB pathway in spotted sea bass ( Lateolabrax maculatus )

doi: 10.3389/fimmu.2023.1181067

Figure Lengend Snippet: Multiple sequence alignment analysis of P2Xs. The N-terminal region, two transmembrane domains (TM1-2), the extracellular loop (ECL), and the C-terminal region of Lm P2Xs are marked above the alignment. Symbol (▴) indicates the conserved cysteine residues. The PKC site, P2X family signature motif, YXXXK motif, and LPS/lipid-binding domain are boxed in orange, pink, red, and green, respectively.

Article Snippet: HEK 293T cells were then transfected with pcDNA3.1- Lm P2Xs or pcDNA3.1 using the PEI 40K Transfection Reagent (Servicebio, China) according to the manufacturer’s instruction.

Techniques: Sequencing, Binding Assay

Journal: Frontiers in Immunology

Article Title: Activated P2X receptors can up-regulate the expressions of inflammation-related genes via NF-κB pathway in spotted sea bass ( Lateolabrax maculatus )

doi: 10.3389/fimmu.2023.1181067

Figure Lengend Snippet: Phylogenetic tree analysis of Lm P2Xs with the selected P2X family members. Phylogenetic tree was constructed using the NJ method within the MEGAX program and run for 10,000 replications. The percentages of bootstrap values for branches are indicated and the Lm P2Xs are shown in bold.

Article Snippet: HEK 293T cells were then transfected with pcDNA3.1- Lm P2Xs or pcDNA3.1 using the PEI 40K Transfection Reagent (Servicebio, China) according to the manufacturer’s instruction.

Techniques: Construct

Journal: Frontiers in Immunology

Article Title: Activated P2X receptors can up-regulate the expressions of inflammation-related genes via NF-κB pathway in spotted sea bass ( Lateolabrax maculatus )

doi: 10.3389/fimmu.2023.1181067

Figure Lengend Snippet: Gene synteny of P2X2 (A) , P2X4/7 (B) , and P2X5 (C) . The Lm P2Xs genome sequence data were obtained from the spotted sea bass genome database ( https://www.ncbi.nlm.nih.gov/-genome/43909 ). Synteny information for other vertebrates was retrieved from the Ensembl database ( http://www.ensembl.org/index.html ). Arrows indicate transcription orientations.

Article Snippet: HEK 293T cells were then transfected with pcDNA3.1- Lm P2Xs or pcDNA3.1 using the PEI 40K Transfection Reagent (Servicebio, China) according to the manufacturer’s instruction.

Techniques: Sequencing

Journal: Frontiers in Immunology

Article Title: Activated P2X receptors can up-regulate the expressions of inflammation-related genes via NF-κB pathway in spotted sea bass ( Lateolabrax maculatus )

doi: 10.3389/fimmu.2023.1181067

Figure Lengend Snippet: Expression analysis of Lm P2Xs in tissues. The expression levels of each gene were normalized to that of EF1α. Data are presented as mean + SEM (N= 4).

Article Snippet: HEK 293T cells were then transfected with pcDNA3.1- Lm P2Xs or pcDNA3.1 using the PEI 40K Transfection Reagent (Servicebio, China) according to the manufacturer’s instruction.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Activated P2X receptors can up-regulate the expressions of inflammation-related genes via NF-κB pathway in spotted sea bass ( Lateolabrax maculatus )

doi: 10.3389/fimmu.2023.1181067

Figure Lengend Snippet: Expressions of Lm P2Xs in primary head kidney leucocytes after stimulation with PGN, LPS, poly (I:C) or ATP. Primary head kidney leukocytes were isolated from the spotted sea bass head kidney and stimulated with PGN (50 μg/mL) LPS (100 μg/mL), LPS (100 μg/mL), Poly (I:C) (50 μg/mL), 100 μM ATP, 1 mM ATP, or PBS (control). The total RNA was extracted for qPCR analysis. The relative expression levels of target genes were normalized to that of EF1α and fold change was obtained by comparing the relative gene expression level of the treated group and control group (defined as 1). The data are shown as mean +SEM (N=3). *P<0.05 and **P<0.01 are considered significant difference.

Article Snippet: HEK 293T cells were then transfected with pcDNA3.1- Lm P2Xs or pcDNA3.1 using the PEI 40K Transfection Reagent (Servicebio, China) according to the manufacturer’s instruction.

Techniques: Isolation, Expressing

Journal: Frontiers in Immunology

Article Title: Activated P2X receptors can up-regulate the expressions of inflammation-related genes via NF-κB pathway in spotted sea bass ( Lateolabrax maculatus )

doi: 10.3389/fimmu.2023.1181067

Figure Lengend Snippet: Effect of activated Lm P2Xs on expressions of pro-inflammatory cytokines (A) and apoptosis-related genes (B) in the HEK 293T cells. HEK 293T cells were transfected with pcDNA3.1- Lm P2Xs or pcDNA3.1. After 24 h, the cells were stimulated with (0.1 mM, 1 mM, or 5 mM) and were collected after 6 h. The empty plasmid transfected cells by PBS stimulation were served as control. The relative expression levels of target genes were normalized to that of βactin and fold change was obtained by comparing the relative gene expression level of the treated group and control group (defined as 1). The data are shown as mean +SEM (N=3). *P<0.05 and **P<0.01 are considered significant difference.

Article Snippet: HEK 293T cells were then transfected with pcDNA3.1- Lm P2Xs or pcDNA3.1 using the PEI 40K Transfection Reagent (Servicebio, China) according to the manufacturer’s instruction.

Techniques: Transfection, Plasmid Preparation, Expressing

Journal: Frontiers in Immunology

Article Title: Activated P2X receptors can up-regulate the expressions of inflammation-related genes via NF-κB pathway in spotted sea bass ( Lateolabrax maculatus )

doi: 10.3389/fimmu.2023.1181067

Figure Lengend Snippet: Effect of activated Lm P2Xs on activity of NF-κB promoter in HEK 293T cells. The pGL4.32 and pRL-TK (negative control) were co-transfected with pcDNA3.1- Lm P2Xs plasmid or pcDNA3.1 plasmid into HEK 293T cells for 24 h. Then the cells were stimulated with ATP (0.1 mM, 1 mM, or 5 mM) or PBS (control) and collected after 6 h. Fold change was obtained by comparing the relative Luciferase activity level of the stimulated group and control group (defined as 1). The data are shown as mean +SEM (N=3). *P<0.05 is considered significant difference.

Article Snippet: HEK 293T cells were then transfected with pcDNA3.1- Lm P2Xs or pcDNA3.1 using the PEI 40K Transfection Reagent (Servicebio, China) according to the manufacturer’s instruction.

Techniques: Activity Assay, Negative Control, Transfection, Plasmid Preparation, Luciferase

Journal: BMC Molecular Biology

Article Title: Sp1/3 and NF-1 mediate basal transcription of the human P2X 1 gene in megakaryoblastic MEG-01 cells

doi: 10.1186/1471-2199-7-10

Figure Lengend Snippet: Sequence of the P2X 1 proximal promoter region . Potential transcription factor binding sites are underlined and in bold. Oligonucleotide sequences used for primer extension analysis (Pet1-4) are indicated by horizontal arrows. End points of plasmid deletion constructs in this region are indicated by "p" and number above the sequence. Sequence numbering is relative to the transcription start site indicated as +1. The translation initiation ATG is boxed. The EMBL/Genbank accession number of this sequence is AJ971536 .

Article Snippet: For transient transfections 5 × 10 4 cells were plated in 96-well plates and incubated with 80 ng of P2X 1 reporter plasmid, 20 ng of pRL-SV40 and 0.3 μl of transfection reagent (GeneJuice™, Novagen Madison, WI).

Techniques: Sequencing, Binding Assay, Plasmid Preparation, Construct

Journal: BMC Molecular Biology

Article Title: Sp1/3 and NF-1 mediate basal transcription of the human P2X 1 gene in megakaryoblastic MEG-01 cells

doi: 10.1186/1471-2199-7-10

Figure Lengend Snippet: The P2X 1 transcription start site is located 365 bp upstream of the start codon . A) 32 P-labelled primers Pet1, Pet2, Pet3 and Pet4 and Con (control primer pTXRHR-100) were annealed to poly(A) RNA from PMA-treated MEG-01 cells and extended with reverse transcriptase. Extension products for Pet1, Pet3 and Pet4 that equate to the same transcription start site are indicated with arrows. (M), molecular mass marker. B) Pet3 extension reaction (lane 6) run in parallel with a sequencing ladder. The location of primers is depicted in Figure 3.

Article Snippet: For transient transfections 5 × 10 4 cells were plated in 96-well plates and incubated with 80 ng of P2X 1 reporter plasmid, 20 ng of pRL-SV40 and 0.3 μl of transfection reagent (GeneJuice™, Novagen Madison, WI).

Techniques: Marker, Sequencing

Journal: BMC Molecular Biology

Article Title: Sp1/3 and NF-1 mediate basal transcription of the human P2X 1 gene in megakaryoblastic MEG-01 cells

doi: 10.1186/1471-2199-7-10

Figure Lengend Snippet: Activity of P2X 1 promoter constructs in transient transfection assays . A) Promoter activity of p-4407/+365 was determined both with (+) and without (-) the prior treatment of cells with 10 nM PMA. Data are means of absolute firefly luciferase activity of three independent transfections. B) Luciferase activity of P2X 1 deletion constructs in PMA treated MEG-01 cells. TSS, transcription start site. PT depicts a polypyrimidine tract located from -1801 to -1405 bp. Values are presented as means of at least three experiments and normalized against the construct p-4407/+365. Names of plasmid constructs correspond to the 5' and 3' ends of the insert relative to the transcription start site e.g. construct p-4407/+365 extends from 4407 bp 5' to 365 bp 3' of the transcription start site.

Article Snippet: For transient transfections 5 × 10 4 cells were plated in 96-well plates and incubated with 80 ng of P2X 1 reporter plasmid, 20 ng of pRL-SV40 and 0.3 μl of transfection reagent (GeneJuice™, Novagen Madison, WI).

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

Journal: BMC Molecular Biology

Article Title: Sp1/3 and NF-1 mediate basal transcription of the human P2X 1 gene in megakaryoblastic MEG-01 cells

doi: 10.1186/1471-2199-7-10

Figure Lengend Snippet: Mutation analysis of transcription factor binding sites . A series of luciferase reporter constructs harbouring site-directed mutations in the NF-κB, NF-1 and Sp1 binding sites of the P2X 1 proximal promoter region were assayed for promoter activity in PMA-treated MEG-01 cells (sequences of mutations are shown in Table 2). Shaded symbols represent mutated binding sites. Values are presented as means ± S.E of at least three independent experiments and are normalized against the wild type p-190/+149 construct for panels A to C and the wild type construct p-4407/+365 in panel D. A) Effects of mutations in the construct p-190/+149. B) Effect of mutation after removal of the potential NF-κB binding site. C) Effect of mutation after removal of the transcription start site that is utilized by smooth muscle cells [15]. Dotted vertical arrow (TSS) represents the transcription start site determined in this study. D) The Sp1a, Sp1b and NF-1 sites were mutated in the original full length 4.7 kb reporter construct p-4407/+365 to generate the triple mutated construct p-4407/+365(mut4). Promoter activities were determined in MEG-01 cells either treated (+) or untreated (-) with PMA.

Article Snippet: For transient transfections 5 × 10 4 cells were plated in 96-well plates and incubated with 80 ng of P2X 1 reporter plasmid, 20 ng of pRL-SV40 and 0.3 μl of transfection reagent (GeneJuice™, Novagen Madison, WI).

Techniques: Mutagenesis, Binding Assay, Luciferase, Construct, Activity Assay

Journal: BMC Molecular Biology

Article Title: Sp1/3 and NF-1 mediate basal transcription of the human P2X 1 gene in megakaryoblastic MEG-01 cells

doi: 10.1186/1471-2199-7-10

Figure Lengend Snippet: Transcription factors bind the endogenous P2X 1 promoter . Formaldehyde cross-linked chromatin was prepared from PMA-treated MEG-01 cells and immunoprecipitated with antibodies to Sp1 (lane 4), Sp3 (lane 5), NF-1 (lane 6), TFIIB (lane 7), or in the absence of antibody (lane 3). PCR was performed with specific primers for the P2X 1 , H4 and Hsp70 promoter and for the GAPDH coding region as a negative control. A sample representative of the total input chromatin (input DNA lane 1) was included in the PCR analysis. Lane 2 shows the supernatant of the 'unbound' sample. PCR products were between 282 and 983 bp in length. PCR cycle numbers were 31 for the P2X 1 , H4 and Hsp70 promoter and 36 for the GAPDH coding region. Primer sequences are given in Table 3.

Article Snippet: For transient transfections 5 × 10 4 cells were plated in 96-well plates and incubated with 80 ng of P2X 1 reporter plasmid, 20 ng of pRL-SV40 and 0.3 μl of transfection reagent (GeneJuice™, Novagen Madison, WI).

Techniques: Immunoprecipitation, Negative Control

Journal: BMC Molecular Biology

Article Title: Sp1/3 and NF-1 mediate basal transcription of the human P2X 1 gene in megakaryoblastic MEG-01 cells

doi: 10.1186/1471-2199-7-10

Figure Lengend Snippet: Cross species conservation of the P2X 1 core promoter . P2X 1 upstream sequence from chimp (chromosome 19 position 4051445–4051944), dog (chromosome 9 position 40295005–40295504), rat (chromosome 10 position 59889637–59890136) and mouse (chromosome 11 position 72611373–72611872) was obtained by BLAST searching of genomic databases via the ENSEMBL genome browser . The 500 bp upstream of the ATG start codon of each gene was aligned using CLUSTAL. Alignment equivalent to -68/+149 of the human P2X 1 core promoter is shown. Sequence identity to the human P2X 1 sequence over this region was 99.1%, 83.4%, 76.8%, and 75.9% for chimp, dog, mouse and rat respectively. "." indicates nucleotides identical to the human P2X 1 sequence. Dashes indicate positions where gaps have been inserted in order to maintain the alignment. The transcription start site of the human P2X 1 gene determined in this study is indicated as +1. Potential transcription start sites predicted by PROSCAN [17] are indicated as arrows. In each species, potential NF-1, Sp1a and Sp1b binding sites (indicated by boxes) are present in equivalent positions to the human P2X 1 core promoter.

Article Snippet: For transient transfections 5 × 10 4 cells were plated in 96-well plates and incubated with 80 ng of P2X 1 reporter plasmid, 20 ng of pRL-SV40 and 0.3 μl of transfection reagent (GeneJuice™, Novagen Madison, WI).

Techniques: Sequencing, Binding Assay

Journal: BMC Molecular Biology

Article Title: Sp1/3 and NF-1 mediate basal transcription of the human P2X 1 gene in megakaryoblastic MEG-01 cells

doi: 10.1186/1471-2199-7-10

Figure Lengend Snippet: Binding site mutants. Positions of mutations in mutant constructs. Schematic illustration of the P2X 1 core promoter mutation constructs. "." indicates positions same as wild type. "~" indicates where sequence has been omitted for clarity.

Article Snippet: For transient transfections 5 × 10 4 cells were plated in 96-well plates and incubated with 80 ng of P2X 1 reporter plasmid, 20 ng of pRL-SV40 and 0.3 μl of transfection reagent (GeneJuice™, Novagen Madison, WI).

Techniques: Binding Assay, Mutagenesis, Construct, Sequencing

Journal: BMC Molecular Biology

Article Title: Sp1/3 and NF-1 mediate basal transcription of the human P2X 1 gene in megakaryoblastic MEG-01 cells

doi: 10.1186/1471-2199-7-10

Figure Lengend Snippet: Oligonucleotides used for ChIP analysis. Primer sequences are 5' to 3'. At the end of oligonucleotide names, f: indicates a sense primer, r: indicates an antisense primer.

Article Snippet: For transient transfections 5 × 10 4 cells were plated in 96-well plates and incubated with 80 ng of P2X 1 reporter plasmid, 20 ng of pRL-SV40 and 0.3 μl of transfection reagent (GeneJuice™, Novagen Madison, WI).

Techniques:

Journal: BMC Molecular Biology

Article Title: Sp1/3 and NF-1 mediate basal transcription of the human P2X 1 gene in megakaryoblastic MEG-01 cells

doi: 10.1186/1471-2199-7-10

Figure Lengend Snippet: Sequence of the P2X 1 proximal promoter region . Potential transcription factor binding sites are underlined and in bold. Oligonucleotide sequences used for primer extension analysis (Pet1-4) are indicated by horizontal arrows. End points of plasmid deletion constructs in this region are indicated by "p" and number above the sequence. Sequence numbering is relative to the transcription start site indicated as +1. The translation initiation ATG is boxed. The EMBL/Genbank accession number of this sequence is AJ971536 .

Article Snippet: A 339 bp fragment (spanning from -190 to +149 bp) was cut from the P2X 1 pGL3-Basic plasmid, cloned into the Mlu I/Hind III sites of pCR-Script™ and used as template for mutagenesis using the QuikChange™ system (Stratagene, La, Jolla CA).

Techniques: Sequencing, Binding Assay, Plasmid Preparation, Construct

Journal: BMC Molecular Biology

Article Title: Sp1/3 and NF-1 mediate basal transcription of the human P2X 1 gene in megakaryoblastic MEG-01 cells

doi: 10.1186/1471-2199-7-10

Figure Lengend Snippet: The P2X 1 transcription start site is located 365 bp upstream of the start codon . A) 32 P-labelled primers Pet1, Pet2, Pet3 and Pet4 and Con (control primer pTXRHR-100) were annealed to poly(A) RNA from PMA-treated MEG-01 cells and extended with reverse transcriptase. Extension products for Pet1, Pet3 and Pet4 that equate to the same transcription start site are indicated with arrows. (M), molecular mass marker. B) Pet3 extension reaction (lane 6) run in parallel with a sequencing ladder. The location of primers is depicted in Figure 3.

Article Snippet: A 339 bp fragment (spanning from -190 to +149 bp) was cut from the P2X 1 pGL3-Basic plasmid, cloned into the Mlu I/Hind III sites of pCR-Script™ and used as template for mutagenesis using the QuikChange™ system (Stratagene, La, Jolla CA).

Techniques: Marker, Sequencing

Journal: BMC Molecular Biology

Article Title: Sp1/3 and NF-1 mediate basal transcription of the human P2X 1 gene in megakaryoblastic MEG-01 cells

doi: 10.1186/1471-2199-7-10

Figure Lengend Snippet: Activity of P2X 1 promoter constructs in transient transfection assays . A) Promoter activity of p-4407/+365 was determined both with (+) and without (-) the prior treatment of cells with 10 nM PMA. Data are means of absolute firefly luciferase activity of three independent transfections. B) Luciferase activity of P2X 1 deletion constructs in PMA treated MEG-01 cells. TSS, transcription start site. PT depicts a polypyrimidine tract located from -1801 to -1405 bp. Values are presented as means of at least three experiments and normalized against the construct p-4407/+365. Names of plasmid constructs correspond to the 5' and 3' ends of the insert relative to the transcription start site e.g. construct p-4407/+365 extends from 4407 bp 5' to 365 bp 3' of the transcription start site.

Article Snippet: A 339 bp fragment (spanning from -190 to +149 bp) was cut from the P2X 1 pGL3-Basic plasmid, cloned into the Mlu I/Hind III sites of pCR-Script™ and used as template for mutagenesis using the QuikChange™ system (Stratagene, La, Jolla CA).

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

Journal: BMC Molecular Biology

Article Title: Sp1/3 and NF-1 mediate basal transcription of the human P2X 1 gene in megakaryoblastic MEG-01 cells

doi: 10.1186/1471-2199-7-10

Figure Lengend Snippet: Mutation analysis of transcription factor binding sites . A series of luciferase reporter constructs harbouring site-directed mutations in the NF-κB, NF-1 and Sp1 binding sites of the P2X 1 proximal promoter region were assayed for promoter activity in PMA-treated MEG-01 cells (sequences of mutations are shown in Table 2). Shaded symbols represent mutated binding sites. Values are presented as means ± S.E of at least three independent experiments and are normalized against the wild type p-190/+149 construct for panels A to C and the wild type construct p-4407/+365 in panel D. A) Effects of mutations in the construct p-190/+149. B) Effect of mutation after removal of the potential NF-κB binding site. C) Effect of mutation after removal of the transcription start site that is utilized by smooth muscle cells [15]. Dotted vertical arrow (TSS) represents the transcription start site determined in this study. D) The Sp1a, Sp1b and NF-1 sites were mutated in the original full length 4.7 kb reporter construct p-4407/+365 to generate the triple mutated construct p-4407/+365(mut4). Promoter activities were determined in MEG-01 cells either treated (+) or untreated (-) with PMA.

Article Snippet: A 339 bp fragment (spanning from -190 to +149 bp) was cut from the P2X 1 pGL3-Basic plasmid, cloned into the Mlu I/Hind III sites of pCR-Script™ and used as template for mutagenesis using the QuikChange™ system (Stratagene, La, Jolla CA).

Techniques: Mutagenesis, Binding Assay, Luciferase, Construct, Activity Assay

Journal: BMC Molecular Biology

Article Title: Sp1/3 and NF-1 mediate basal transcription of the human P2X 1 gene in megakaryoblastic MEG-01 cells

doi: 10.1186/1471-2199-7-10

Figure Lengend Snippet: Transcription factors bind the endogenous P2X 1 promoter . Formaldehyde cross-linked chromatin was prepared from PMA-treated MEG-01 cells and immunoprecipitated with antibodies to Sp1 (lane 4), Sp3 (lane 5), NF-1 (lane 6), TFIIB (lane 7), or in the absence of antibody (lane 3). PCR was performed with specific primers for the P2X 1 , H4 and Hsp70 promoter and for the GAPDH coding region as a negative control. A sample representative of the total input chromatin (input DNA lane 1) was included in the PCR analysis. Lane 2 shows the supernatant of the 'unbound' sample. PCR products were between 282 and 983 bp in length. PCR cycle numbers were 31 for the P2X 1 , H4 and Hsp70 promoter and 36 for the GAPDH coding region. Primer sequences are given in Table 3.

Article Snippet: A 339 bp fragment (spanning from -190 to +149 bp) was cut from the P2X 1 pGL3-Basic plasmid, cloned into the Mlu I/Hind III sites of pCR-Script™ and used as template for mutagenesis using the QuikChange™ system (Stratagene, La, Jolla CA).

Techniques: Immunoprecipitation, Negative Control

Journal: BMC Molecular Biology

Article Title: Sp1/3 and NF-1 mediate basal transcription of the human P2X 1 gene in megakaryoblastic MEG-01 cells

doi: 10.1186/1471-2199-7-10

Figure Lengend Snippet: Cross species conservation of the P2X 1 core promoter . P2X 1 upstream sequence from chimp (chromosome 19 position 4051445–4051944), dog (chromosome 9 position 40295005–40295504), rat (chromosome 10 position 59889637–59890136) and mouse (chromosome 11 position 72611373–72611872) was obtained by BLAST searching of genomic databases via the ENSEMBL genome browser . The 500 bp upstream of the ATG start codon of each gene was aligned using CLUSTAL. Alignment equivalent to -68/+149 of the human P2X 1 core promoter is shown. Sequence identity to the human P2X 1 sequence over this region was 99.1%, 83.4%, 76.8%, and 75.9% for chimp, dog, mouse and rat respectively. "." indicates nucleotides identical to the human P2X 1 sequence. Dashes indicate positions where gaps have been inserted in order to maintain the alignment. The transcription start site of the human P2X 1 gene determined in this study is indicated as +1. Potential transcription start sites predicted by PROSCAN [17] are indicated as arrows. In each species, potential NF-1, Sp1a and Sp1b binding sites (indicated by boxes) are present in equivalent positions to the human P2X 1 core promoter.

Article Snippet: A 339 bp fragment (spanning from -190 to +149 bp) was cut from the P2X 1 pGL3-Basic plasmid, cloned into the Mlu I/Hind III sites of pCR-Script™ and used as template for mutagenesis using the QuikChange™ system (Stratagene, La, Jolla CA).

Techniques: Sequencing, Binding Assay

Journal: BMC Molecular Biology

Article Title: Sp1/3 and NF-1 mediate basal transcription of the human P2X 1 gene in megakaryoblastic MEG-01 cells

doi: 10.1186/1471-2199-7-10

Figure Lengend Snippet: Binding site mutants. Positions of mutations in mutant constructs. Schematic illustration of the P2X 1 core promoter mutation constructs. "." indicates positions same as wild type. "~" indicates where sequence has been omitted for clarity.

Article Snippet: A 339 bp fragment (spanning from -190 to +149 bp) was cut from the P2X 1 pGL3-Basic plasmid, cloned into the Mlu I/Hind III sites of pCR-Script™ and used as template for mutagenesis using the QuikChange™ system (Stratagene, La, Jolla CA).

Techniques: Binding Assay, Mutagenesis, Construct, Sequencing

Journal: BMC Molecular Biology

Article Title: Sp1/3 and NF-1 mediate basal transcription of the human P2X 1 gene in megakaryoblastic MEG-01 cells

doi: 10.1186/1471-2199-7-10

Figure Lengend Snippet: Oligonucleotides used for ChIP analysis. Primer sequences are 5' to 3'. At the end of oligonucleotide names, f: indicates a sense primer, r: indicates an antisense primer.

Article Snippet: A 339 bp fragment (spanning from -190 to +149 bp) was cut from the P2X 1 pGL3-Basic plasmid, cloned into the Mlu I/Hind III sites of pCR-Script™ and used as template for mutagenesis using the QuikChange™ system (Stratagene, La, Jolla CA).

Techniques:

Journal: PLoS ONE

Article Title: Sensory Dysfunction of Bladder Mucosa and Bladder Oversensitivity in a Rat Model of Metabolic Syndrome

doi: 10.1371/journal.pone.0045578

Figure Lengend Snippet: Western blot analysis with specific antibodies to the TRPV1 receptor, purinergic P2X 2 receptor, and P2X 3 receptor of the rat mucosal layer and the purinergic P2X 1 receptor of the rat smooth muscle layer in controls and FFRs with different in cystometric presentations. A. TRPV1 receptor: The TRPV1 antibody produced a clear single band at 95kDa. B. Purinergic P2X 2 receptor C. Purinergic P2X 3 mature receptor: the predominant P2X 3 form (65kDa). D. Up-regulation of P2X3 native and intermediate polypeptides (up to 55kD) were shown in both FFR groups. E. Purinergic P2X 1 receptor Experiments were repeated two times and representative blots are shown. Data of proteins expression (ratios of signal intensities of investigated receptors relative to β-actin or GAPDH) were calculated with 8 samples in each group. These data of Mean ± SE were standardized and expressed in percentage in which the value of the control group is treated as 100%. Theses values were shown in the bar graph. An asterisk indicates a significant difference between controls and FFR groups (One-way ANOVA with Dunnett’s test, p<0.05).

Article Snippet: After performing control cystometrographies with saline infusion, rats were subjected to one of four types of physiological experiments: (a) repeatable responses to acidic ATP solution stimulation; (b) capsaicin pretreated to inhibit the response of acidic ATP solution; (c) intravesical administration of liposome (2 mg/ml; Lipella Pharmaceuticals, Pittsburgh, Pennsylvania), a mucosal protective agent , , to partially normalize stimulation by acidic ATP solution; or (d) intravenous administration of pyridoxal 5-phosphate 6-azophenyl-2′,4′-disulfonic acid (PPADS, 70 µmol/kg; Sigma), a commonly used noncompetitively antagonist for heteromeric P2X 2 /P2X 3 , homomeric P2X 3 and P2X 1 receptors , to reverse the stimulation by acidic ATP solution.

Techniques: Western Blot, Produced, Expressing

Journal: BMC Immunology

Article Title: Expression of P2 receptors in human B cells and Epstein-Barr virus-transformed lymphoblastoid cell lines

doi: 10.1186/1471-2172-7-22

Figure Lengend Snippet: Relative expression of P2 receptors in B cells . The relative expression of the P2X receptor gene (upper; P2X 1 , P2X 2 , P2X 3 , P2X 4 , P2X 5 , P2X 6 , and P2X 7 ) and P2Y receptor gene (lower; P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , P2Y 13 , and P2Y 14 ) in B cells are presented (n = 4). The expression was normalized to GAPDH. P2X receptors were calibrated by P2X 1 and P2Y receptors were calibrated by P2Y 1 . Data are the mean ± SEM.

Article Snippet: The membrane was incubated with rabbit polyclonal antibodies (Alomone Labs, Jerusalem, Israel) against P2X 1 receptor, P2X 4 receptor, P2X 7 receptor, P2Y 1 receptor, or P2Y 11 receptor in TBST for 2 hours at room temperature, washed with TBST, and incubated with secondary anti-rabbit IgG (Amersham Pharmacia Biotech) in TBST for 1 hour.

Techniques: Expressing

Journal: BMC Immunology

Article Title: Expression of P2 receptors in human B cells and Epstein-Barr virus-transformed lymphoblastoid cell lines

doi: 10.1186/1471-2172-7-22

Figure Lengend Snippet: Comparison of relative P2 receptor expression in B cells and LCLs . The relative expression of the P2X receptor gene (upper) and P2Y receptor gene (lower) in B cells, EBV-infected B cells for 2 weeks, and LCLs are presented (n = 4). The expression was normalized to GAPDH. P2X receptors were calibrated by P2X 1 of B cells and P2Y receptors were calibrated by P2Y 1 of B cells. Data are the mean ± SEM.

Article Snippet: The membrane was incubated with rabbit polyclonal antibodies (Alomone Labs, Jerusalem, Israel) against P2X 1 receptor, P2X 4 receptor, P2X 7 receptor, P2Y 1 receptor, or P2Y 11 receptor in TBST for 2 hours at room temperature, washed with TBST, and incubated with secondary anti-rabbit IgG (Amersham Pharmacia Biotech) in TBST for 1 hour.

Techniques: Expressing, Infection

Journal: BMC Immunology

Article Title: Expression of P2 receptors in human B cells and Epstein-Barr virus-transformed lymphoblastoid cell lines

doi: 10.1186/1471-2172-7-22

Figure Lengend Snippet: Relative expression of P2 receptors in PBMCs . The relative expression of the P2X receptor gene (upper) and P2Y receptor gene (lower) in PBMCs are presented (n = 4). The expression was normalized to GAPDH. P2X receptors were calibrated by P2X 1 and P2Y receptors were calibrated by P2Y 1 . Data are the mean ± SEM.

Article Snippet: The membrane was incubated with rabbit polyclonal antibodies (Alomone Labs, Jerusalem, Israel) against P2X 1 receptor, P2X 4 receptor, P2X 7 receptor, P2Y 1 receptor, or P2Y 11 receptor in TBST for 2 hours at room temperature, washed with TBST, and incubated with secondary anti-rabbit IgG (Amersham Pharmacia Biotech) in TBST for 1 hour.

Techniques: Expressing

Journal: BMC Immunology

Article Title: Expression of P2 receptors in human B cells and Epstein-Barr virus-transformed lymphoblastoid cell lines

doi: 10.1186/1471-2172-7-22

Figure Lengend Snippet: Relative expression of P2 receptors in LCLs . The relative expression of the P2X receptor gene (upper) and P2Y receptor gene (lower) in EBV-infected B cells (2 wks), LCLs, are presented (n = 4). The expression was normalized to GAPDH. P2X receptors were calibrated by P2X 1 and P2Y receptors were calibrated by P2Y 1 . Data are the mean ± SEM.

Article Snippet: The membrane was incubated with rabbit polyclonal antibodies (Alomone Labs, Jerusalem, Israel) against P2X 1 receptor, P2X 4 receptor, P2X 7 receptor, P2Y 1 receptor, or P2Y 11 receptor in TBST for 2 hours at room temperature, washed with TBST, and incubated with secondary anti-rabbit IgG (Amersham Pharmacia Biotech) in TBST for 1 hour.

Techniques: Expressing, Infection

Journal: BMC Immunology

Article Title: Expression of P2 receptors in human B cells and Epstein-Barr virus-transformed lymphoblastoid cell lines

doi: 10.1186/1471-2172-7-22

Figure Lengend Snippet: P2 receptors (P2X 1 , P2X 4 , P2X 7 , P2Y 1 , and P2Y 11 ) compared by Western blotting in B cells and LCLs . Proteins extracted from B cells and LCLs were probed with rabbit polyclonal antibodies directed against P2X 1 (60-kDa), P2X 4 (65-kDa), P2X 7 (68-kDa), P2Y 1 (66-kDa), and P2Y 11 (50-kDa) (left). Membranes were re-probed by GAPDH (40-kDa) (right). Data are representatives of 4 independent experiments.

Article Snippet: The membrane was incubated with rabbit polyclonal antibodies (Alomone Labs, Jerusalem, Israel) against P2X 1 receptor, P2X 4 receptor, P2X 7 receptor, P2Y 1 receptor, or P2Y 11 receptor in TBST for 2 hours at room temperature, washed with TBST, and incubated with secondary anti-rabbit IgG (Amersham Pharmacia Biotech) in TBST for 1 hour.

Techniques: Western Blot

Journal: BMC Immunology

Article Title: Expression of P2 receptors in human B cells and Epstein-Barr virus-transformed lymphoblastoid cell lines

doi: 10.1186/1471-2172-7-22

Figure Lengend Snippet: Sequence details for all P2X and P2Y receptor subtypes and reference (GAPDH) primers.

Article Snippet: The membrane was incubated with rabbit polyclonal antibodies (Alomone Labs, Jerusalem, Israel) against P2X 1 receptor, P2X 4 receptor, P2X 7 receptor, P2Y 1 receptor, or P2Y 11 receptor in TBST for 2 hours at room temperature, washed with TBST, and incubated with secondary anti-rabbit IgG (Amersham Pharmacia Biotech) in TBST for 1 hour.

Techniques: Sequencing