Journal: Nucleic Acids Research

Article Title: Activation of APE1/Ref-1 is dependent on reactive oxygen species generated after purinergic receptor stimulation by ATP

doi: 10.1093/nar/gki751

Figure Lengend Snippet: Purinergic triggering causes APE1/Ref-1 cytoplasm to nucleus translocation and its maintenance in the nuclear compartment in a protein-neosynthesis independent manner. ( A ) ARO cells were cultured in serum-free medium for 48 h and then stimulated or not with ATP for the indicated times and in the presence of the protein synthesis inhibitor CHX (15 μg/ml) 5 min before and during the ATP treatment. After stimulation, nuclear and cytoplasmic extracts were prepared and analysed by western blot analysis for the presence of APE1/Ref-1, as described above. Western blot shown is representative of three. Lower panel, values obtained from densitometric analysis of three independent western blot experiments were reported as histograms. Columns and bars indicate the means ± SD from three independent experiments. Black bars represent the nuclear levels of APE1/Ref-1 and the white dotted bars represent the cytoplasmic APE1/Ref-1 protein levels obtained from densitometric analysis of three independent experiments, normalized versus actin ( * , p ≤ 0.05; ** , p < 0.001 compared with controls). ( B ) ARO cells were treated as in (A). Cells were then fixed and stained for APE1/Ref-1 (green). Nuclei were visualized by propidium iodide counterstaining (red). ( C ). P2 receptor triggering is responsible for APE1/Ref-1 cytoplasm to nucleus translocation induced by ATP treatment. ARO cells were cultured in serum-free medium for 48 h and then stimulated with the indicated amounts of ATP for 10 min. Representative western blot analysis, performed as described in Materials and Methods, of APE1/Ref-1 expression levels of both nuclear and cytoplasmic extracts of ARO cells stimulated or not with decreasing amounts of ATP, as indicated (lanes 2–4). In order to test whether extracellular ATP activates APE1/Ref-1 translocation by activation of P2 cell-surface purinergic receptors, ARO cells were pre-treated with the specific P2 receptor antagonist suramin (100 μM) 30 min before the addition of 100 μM ATP (lanes 5 and 6), or with ATP-γS (lane 7) or with adenosine (lane 8) alone. ( D ). Stimulation of APE1/Ref-1 nuclear translocation by P2Y triggering in ARO cells. ARO cells were cultured in serum-free medium for 48 h and then stimulated with the indicated agonists. Representative western blot analysis, performed as described in Materials and Methods, of APE1/Ref-1 expression levels of nuclear and cytoplasmic extracts of ARO cells is reported. ( E ). P2 receptors expression by ARO cells. Left panels: PCR amplification of cDNAs with P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 and GAPDH primer pairs. cDNAs were synthesized from RNA template prepared from ARO cells or MG-63 cells, as a positive control. As indicated, the bands of 259 (lane 1, upper part), 362 (lane 2, upper part), 449 and 530 bp (lanes 2, lower part) correspond to amplification products specific for P2Y 1 and P2Y 2 , P2Y 4 and P2Y 6 receptors cDNA, respectively. As a control of cDNA quality, GAPDH yields a larger 598 bp band. The control reactions contained not retro-transcribed mRNA instead of template cDNA (lanes 3 and 4). Right panel: western blot analysis of P2Y 1 and P2Y 2 receptors in ARO cells. Analysis was performed as described in Materials and Methods. To assay for the specificity of the recognized bands, competition experiments with the specific peptides of the two receptors were performed by pre-incubating each antibody for 30 min at room temperature with each specific peptide (lane 3). ( F ). Continuous purinergic triggering controls the subcellular levels of APE1/Ref-1 in ARO cells. ARO cells were treated with the indicated amounts of the ATP/ADPase apyrase and with the P2 receptor inhibitor suramin for the indicated times. Representative western blot analysis, performed as described in Materials and Methods, of APE1/Ref-1 expression levels of nuclear and cytoplasmic extracts of ARO cells is reported.

Article Snippet: Then, 50 μg of extracts were separated onto a 10% SDS–PAGE, blotted onto nitrocellulose membranes and assayed for the presence of P2Y 1 and P2Y 2 proteins by using specific polyclonal antibodies (Alomone Labs., Jerusalem, Israel).

Techniques: Translocation Assay, Cell Culture, Western Blot, Staining, Expressing, Activation Assay, Amplification, Synthesized, Positive Control

Journal: BMC Research Notes

Article Title: Immunogold electron microscopic evidence of in situ formation of homo- and heteromeric purinergic adenosine A 1 and P2Y 2 receptors in rat brain

doi: 10.1186/1756-0500-3-323

Figure Lengend Snippet: Bar graphs comparing the relative distributions of A 1 R(A1)- and P2Y 2 R(Y2)-immunoreactive elements in each brain region (A-C) and in transfected HEK293T cells (D) . The P2Y 2 R-P2Y 2 R, A 1 R-A 1 R and A 1 R-P2Y 2 R dimers are indicated by Y2-Y2, A1-A1 and A1-Y2, respectively. Total number of immunoreactive gold particles on the cell surface was defined as 100%. Each column represents the average frequency (± SD) from three cells. Raw data are shown in the tables under the graphs. Data are means of three independent experiments.

Article Snippet: Cells were washed and then stained with Alexa 568-conjugated goat anti-rat IgG antibody (1:200, Invitrogen, Carlsbad, CA) for A 1 R or Alexa 488-conjugated goat anti-mouse IgG antibody (1:200, Invitrogen) for P2Y 2 R. The characterization of antibodies for rat brain sections was previously reported, although the rabbit polyclonal anti-P2Y 2 R antibody (anti-P2Y 2 R; 1 μg/ml, Alomone Labs, Jerusalem, Israel) was used instead of the rabbit polyclonal anti-P2Y 1 R antibody [ , ].

Techniques: Transfection

Journal: BMC Research Notes

Article Title: Immunogold electron microscopic evidence of in situ formation of homo- and heteromeric purinergic adenosine A 1 and P2Y 2 receptors in rat brain

doi: 10.1186/1756-0500-3-323

Figure Lengend Snippet: Co-localization of A 1 R and P2Y 2 R . A-C . Confocal images of double immunostained Myc-P2Y 2 R (A; green), HA-A 1 R (B; red), and their merge (C; yellow) in co-transfected HEK293T cells. The co-localization of HA-A 1 R and Myc-P2Y 2 R is evident at the cell surface membrane (small arrow). D-L . Confocal images of double immunofluorescence staining in several rat brain regions. P2Y 2 R (D, G, J; red) and A 1 R (E, H, K; green) immunoreactivities were detected in Purkinje cells (D-F), cerebellar nuclei (G-I), and hippocampal CA3 pyramidal cells (J-L). Co-localizations of A 1 R and P2Y 2 R (F, I, L; yellow) were detected in the soma (large arrows) of all tissues, in dendrites of the Purkinje cells, and in neurons of the cerebellar nuclei (arrowheads). Yellow bar indicates 500 μm (A-C) and white bar indicates 100 μm (D-L). Mol: cerebellar molecular layer, Gr: cerebellar granule cell layer. Fluorescent images were collected via confocal laser scanning microscopy (Zeiss LSM410, Carl Zeiss, Oberkochen, Germany) each 10-μm optical slice consisted of a stack of 20 0.5-μm thick sections. Serial optical sections were recorded using an air objective lens of (40×, numerical aperture; 0.6).

Article Snippet: Cells were washed and then stained with Alexa 568-conjugated goat anti-rat IgG antibody (1:200, Invitrogen, Carlsbad, CA) for A 1 R or Alexa 488-conjugated goat anti-mouse IgG antibody (1:200, Invitrogen) for P2Y 2 R. The characterization of antibodies for rat brain sections was previously reported, although the rabbit polyclonal anti-P2Y 2 R antibody (anti-P2Y 2 R; 1 μg/ml, Alomone Labs, Jerusalem, Israel) was used instead of the rabbit polyclonal anti-P2Y 1 R antibody [ , ].

Techniques: Transfection, Double Immunofluorescence Staining, Confocal Laser Scanning Microscopy

Journal: BMC Research Notes

Article Title: Immunogold electron microscopic evidence of in situ formation of homo- and heteromeric purinergic adenosine A 1 and P2Y 2 receptors in rat brain

doi: 10.1186/1756-0500-3-323

Figure Lengend Snippet: Co-immunoprecipitation of A 1 R from cerebral cortex (lane 1), cerebellum (lane 2), and hippocampus (lane 3) . Immunoblotting analyses of extracts of rat brain with anti-A 1 R (A), anti-P2Y 2 R (B), and anti-P2Y 2 R with the control peptide of P2Y 2 R (C). Membrane extracts from each region were immunoprecipitated with anti-A 1 R, and analyzed by immunoblotting with anti-P2Y 2 R (D), anti-P2Y 2 R with the control peptide of P2Y 2 R (E), anti-A 1 R (F). No bands were seen in C, E demonstrating the specificity of the antibodies. It was confirmed that immunoprecpitation without primary antibody resulted in no detectable receptor bands in the immunoblotting (data not shown). Approximate molecular masses are shown in kDa.

Article Snippet: Cells were washed and then stained with Alexa 568-conjugated goat anti-rat IgG antibody (1:200, Invitrogen, Carlsbad, CA) for A 1 R or Alexa 488-conjugated goat anti-mouse IgG antibody (1:200, Invitrogen) for P2Y 2 R. The characterization of antibodies for rat brain sections was previously reported, although the rabbit polyclonal anti-P2Y 2 R antibody (anti-P2Y 2 R; 1 μg/ml, Alomone Labs, Jerusalem, Israel) was used instead of the rabbit polyclonal anti-P2Y 1 R antibody [ , ].

Techniques: Immunoprecipitation, Western Blot

Journal: BMC Research Notes

Article Title: Immunogold electron microscopic evidence of in situ formation of homo- and heteromeric purinergic adenosine A 1 and P2Y 2 receptors in rat brain

doi: 10.1186/1756-0500-3-323

Figure Lengend Snippet: Immunogold electron microscopy of A 1 R and P2Y 2 R visualized using nanogold particles in transfected HEK293T cells (A-D) and rat brain (E-G) . A: Localization of HA-A 1 R (large particles) detected with anti-HA in HA-A 1 R-transfected HEK293T cells. B: Localization of Myc-P2Y 2 R (small particles) detected with anti-Myc in Myc-P2Y 2 R-transfected HEK293T cells. C: Anti-HA and anti-Myc immuno-localization of HA-A 1 R and Myc-P2Y 2 R in co-transfected HEK293T cells. D: HA-A 1 R-transfected HEK293T cells incubated with both anti-HA and anti-Myc. E-G: Localization of A 1 R and P2Y 2 R in cortical pyramidal cells (E), Purkinje cells (F), and hippocampal pyramidal cells (G) detected with both anti-A 1 R and anti-P2Y 2 R. Arrows indicate two adjacent receptors on the cell membrane. Bars represent 100 nm. CM, cell membrane; CP, cytoplasm.

Article Snippet: Cells were washed and then stained with Alexa 568-conjugated goat anti-rat IgG antibody (1:200, Invitrogen, Carlsbad, CA) for A 1 R or Alexa 488-conjugated goat anti-mouse IgG antibody (1:200, Invitrogen) for P2Y 2 R. The characterization of antibodies for rat brain sections was previously reported, although the rabbit polyclonal anti-P2Y 2 R antibody (anti-P2Y 2 R; 1 μg/ml, Alomone Labs, Jerusalem, Israel) was used instead of the rabbit polyclonal anti-P2Y 1 R antibody [ , ].

Techniques: Electron Microscopy, Transfection, Incubation

Journal: BMC Research Notes

Article Title: Immunogold electron microscopic evidence of in situ formation of homo- and heteromeric purinergic adenosine A 1 and P2Y 2 receptors in rat brain

doi: 10.1186/1756-0500-3-323

Figure Lengend Snippet: Bar graphs comparing the relative distributions of A 1 R(A1)- and P2Y 2 R(Y2)-immunoreactive elements in each brain region (A-C) and in transfected HEK293T cells (D) . The P2Y 2 R-P2Y 2 R, A 1 R-A 1 R and A 1 R-P2Y 2 R dimers are indicated by Y2-Y2, A1-A1 and A1-Y2, respectively. Total number of immunoreactive gold particles on the cell surface was defined as 100%. Each column represents the average frequency (± SD) from three cells. Raw data are shown in the tables under the graphs. Data are means of three independent experiments.

Article Snippet: Cells were washed and then stained with Alexa 568-conjugated goat anti-rat IgG antibody (1:200, Invitrogen, Carlsbad, CA) for A 1 R or Alexa 488-conjugated goat anti-mouse IgG antibody (1:200, Invitrogen) for P2Y 2 R. The characterization of antibodies for rat brain sections was previously reported, although the rabbit polyclonal anti-P2Y 2 R antibody (anti-P2Y 2 R; 1 μg/ml, Alomone Labs, Jerusalem, Israel) was used instead of the rabbit polyclonal anti-P2Y 1 R antibody [ , ].

Techniques: Transfection

Journal: BMC Research Notes

Article Title: Immunogold electron microscopic evidence of in situ formation of homo- and heteromeric purinergic adenosine A 1 and P2Y 2 receptors in rat brain

doi: 10.1186/1756-0500-3-323

Figure Lengend Snippet: Co-localization of A 1 R and P2Y 2 R . A-C . Confocal images of double immunostained Myc-P2Y 2 R (A; green), HA-A 1 R (B; red), and their merge (C; yellow) in co-transfected HEK293T cells. The co-localization of HA-A 1 R and Myc-P2Y 2 R is evident at the cell surface membrane (small arrow). D-L . Confocal images of double immunofluorescence staining in several rat brain regions. P2Y 2 R (D, G, J; red) and A 1 R (E, H, K; green) immunoreactivities were detected in Purkinje cells (D-F), cerebellar nuclei (G-I), and hippocampal CA3 pyramidal cells (J-L). Co-localizations of A 1 R and P2Y 2 R (F, I, L; yellow) were detected in the soma (large arrows) of all tissues, in dendrites of the Purkinje cells, and in neurons of the cerebellar nuclei (arrowheads). Yellow bar indicates 500 μm (A-C) and white bar indicates 100 μm (D-L). Mol: cerebellar molecular layer, Gr: cerebellar granule cell layer. Fluorescent images were collected via confocal laser scanning microscopy (Zeiss LSM410, Carl Zeiss, Oberkochen, Germany) each 10-μm optical slice consisted of a stack of 20 0.5-μm thick sections. Serial optical sections were recorded using an air objective lens of (40×, numerical aperture; 0.6).

Article Snippet: Cells were washed and then stained with Alexa 568-conjugated goat anti-rat IgG antibody (1:200, Invitrogen, Carlsbad, CA) for A 1 R or Alexa 488-conjugated goat anti-mouse IgG antibody (1:200, Invitrogen) for P2Y 2 R. The characterization of antibodies for rat brain sections was previously reported, although the rabbit polyclonal anti-P2Y 2 R antibody (anti-P2Y 2 R; 1 μg/ml, Alomone Labs, Jerusalem, Israel) was used instead of the rabbit polyclonal anti-P2Y 1 R antibody [ , ].

Techniques: Transfection, Double Immunofluorescence Staining, Confocal Laser Scanning Microscopy

Journal: BMC Research Notes

Article Title: Immunogold electron microscopic evidence of in situ formation of homo- and heteromeric purinergic adenosine A 1 and P2Y 2 receptors in rat brain

doi: 10.1186/1756-0500-3-323

Figure Lengend Snippet: Co-immunoprecipitation of A 1 R from cerebral cortex (lane 1), cerebellum (lane 2), and hippocampus (lane 3) . Immunoblotting analyses of extracts of rat brain with anti-A 1 R (A), anti-P2Y 2 R (B), and anti-P2Y 2 R with the control peptide of P2Y 2 R (C). Membrane extracts from each region were immunoprecipitated with anti-A 1 R, and analyzed by immunoblotting with anti-P2Y 2 R (D), anti-P2Y 2 R with the control peptide of P2Y 2 R (E), anti-A 1 R (F). No bands were seen in C, E demonstrating the specificity of the antibodies. It was confirmed that immunoprecpitation without primary antibody resulted in no detectable receptor bands in the immunoblotting (data not shown). Approximate molecular masses are shown in kDa.

Article Snippet: Cells were washed and then stained with Alexa 568-conjugated goat anti-rat IgG antibody (1:200, Invitrogen, Carlsbad, CA) for A 1 R or Alexa 488-conjugated goat anti-mouse IgG antibody (1:200, Invitrogen) for P2Y 2 R. The characterization of antibodies for rat brain sections was previously reported, although the rabbit polyclonal anti-P2Y 2 R antibody (anti-P2Y 2 R; 1 μg/ml, Alomone Labs, Jerusalem, Israel) was used instead of the rabbit polyclonal anti-P2Y 1 R antibody [ , ].

Techniques: Immunoprecipitation, Western Blot

Journal: BMC Research Notes

Article Title: Immunogold electron microscopic evidence of in situ formation of homo- and heteromeric purinergic adenosine A 1 and P2Y 2 receptors in rat brain

doi: 10.1186/1756-0500-3-323

Figure Lengend Snippet: Immunogold electron microscopy of A 1 R and P2Y 2 R visualized using nanogold particles in transfected HEK293T cells (A-D) and rat brain (E-G) . A: Localization of HA-A 1 R (large particles) detected with anti-HA in HA-A 1 R-transfected HEK293T cells. B: Localization of Myc-P2Y 2 R (small particles) detected with anti-Myc in Myc-P2Y 2 R-transfected HEK293T cells. C: Anti-HA and anti-Myc immuno-localization of HA-A 1 R and Myc-P2Y 2 R in co-transfected HEK293T cells. D: HA-A 1 R-transfected HEK293T cells incubated with both anti-HA and anti-Myc. E-G: Localization of A 1 R and P2Y 2 R in cortical pyramidal cells (E), Purkinje cells (F), and hippocampal pyramidal cells (G) detected with both anti-A 1 R and anti-P2Y 2 R. Arrows indicate two adjacent receptors on the cell membrane. Bars represent 100 nm. CM, cell membrane; CP, cytoplasm.

Article Snippet: Cells were washed and then stained with Alexa 568-conjugated goat anti-rat IgG antibody (1:200, Invitrogen, Carlsbad, CA) for A 1 R or Alexa 488-conjugated goat anti-mouse IgG antibody (1:200, Invitrogen) for P2Y 2 R. The characterization of antibodies for rat brain sections was previously reported, although the rabbit polyclonal anti-P2Y 2 R antibody (anti-P2Y 2 R; 1 μg/ml, Alomone Labs, Jerusalem, Israel) was used instead of the rabbit polyclonal anti-P2Y 1 R antibody [ , ].

Techniques: Electron Microscopy, Transfection, Incubation

Journal: Stem Cells International

Article Title: Purinergic Signaling Pathway in Human Olfactory Neuronal Precursor Cells

doi: 10.1155/2019/2728786

Figure Lengend Snippet: Detection of P2 receptors in the clonal culture of the hOE. Cloned cells were scraped with a RIPA buffer, and cell lysates were assayed by Western blot to immunodetect P2 receptor subtypes. Representative chemiluminescent bands corresponding to P2X receptors are shown in (a) and to P2Y receptors in (b). (c) shows antibody specificity controls assessed by omission of primary antibody (P2X3) or by preadsorption of anti-P2X5 and anti-P2Y2 with the corresponding blocking peptides. The molecular weights of sample bands were corroborated using biotinylated molecular weight standards. (d) shows immunodetection of P2Y2 and P2Y4 receptors in both the fresh exfoliate (FE) sample and the clonal culture (CC) from the hOE.

Article Snippet: Antibody specificity controls were assessed either without incubation with the primary antibody (P2X3) or by preadsorption of primary antibodies with the corresponding blocking peptides (control antigens for APR-027 (P2X5) and for APR-010 (P2Y2); Alomone), for those P2 receptors that revealed more than one chemiluminescent band.

Techniques: Clone Assay, Western Blot, Blocking Assay, Molecular Weight, Immunodetection

Journal: Stem Cells International

Article Title: Purinergic Signaling Pathway in Human Olfactory Neuronal Precursor Cells

doi: 10.1155/2019/2728786

Figure Lengend Snippet: Detection of P2 receptors in the clonal culture of the hOE. Cloned cells were scraped with a RIPA buffer, and cell lysates were assayed by Western blot to immunodetect P2 receptor subtypes. Representative chemiluminescent bands corresponding to P2X receptors are shown in (a) and to P2Y receptors in (b). (c) shows antibody specificity controls assessed by omission of primary antibody (P2X3) or by preadsorption of anti-P2X5 and anti-P2Y2 with the corresponding blocking peptides. The molecular weights of sample bands were corroborated using biotinylated molecular weight standards. (d) shows immunodetection of P2Y2 and P2Y4 receptors in both the fresh exfoliate (FE) sample and the clonal culture (CC) from the hOE.

Article Snippet: Antibody specificity controls were assessed either without incubation with the primary antibody (P2X3) or by preadsorption of primary antibodies with the corresponding blocking peptides (control antigens for APR-027 (P2X5) and for APR-010 (P2Y2); Alomone), for those P2 receptors that revealed more than one chemiluminescent band.

Techniques: Clone Assay, Western Blot, Blocking Assay, Molecular Weight, Immunodetection