Journal: bioRxiv

Article Title: EROS-mediated control of NOX2 and P2X7 biosynthesis

doi: 10.1101/2021.09.14.460103

Figure Lengend Snippet: (A-C) P2X7 expression analysed by western blotting of macrophages isolated from control, EROS -/- (A) and gp91 phox -/- mice (B) and of control PLB985 cells and an EROS knock-out clone (C). (D-E) P2X7 expression in RAW264.7 cells overexpressing a FLAG-tagged EROS vector (D) and in HEK293 cells transiently expressing the specified constructs (E). (F-G) Interaction between EROS and P2X7 probed by immunoprecipitation of EROS from RAW264.7 EROS-FLAG macrophages followed by immunoblot for P2X7 (F) and by NanoBIT assay in live HEK293 cells expressing the LgBIT-fused EROS vector with a SmBIT-fused P2X7 vector (G). (H) P2X1 expression in macrophages isolated from EROS -/- mice compared to control. n= 5 biological replicates. (I) P2X1 abundance upon co-transfection with EROS construct in HEK293 cells. n= representative of 3 independent experiments. See also Figure S3.

Article Snippet: The following plasmids were obtained from Origene: mouse gp91 phox /nox2/cybb (MC204867), mouse GFP-tagged gp91 phox /nox2/cybb (MG208975), mouse EROS/cybc1 (MC201263), human EROS/CYBC1 (SC324452), human gp91 phox /CYBB (SC122091), human p22 phox /CYBA (SC101113), human NOX4 (SC322623), mouse GFP-tagged P2X7 (MR227216), human P2X1 (SC118594) and human P2X4 (SC122124).

Techniques: Expressing, Western Blot, Isolation, Knock-Out, Plasmid Preparation, Construct, Immunoprecipitation, Cotransfection

Journal: Purinergic Signalling

Article Title: Diadenosine pentaphosphate regulates dendrite growth and number in cultured hippocampal neurons

doi: 10.1007/s11302-023-09944-z

Figure Lengend Snippet: Purinergic receptors P2X1 regulate dendrite growth and number in cultured hippocampal neurons. A Hippocampal neurons cultured for 3 DIV stained with antibodies against β-III-tubulin (red channel) and P2X1 (green channel). Scale bar: 50 μm. B Hippocampal neurons cultured for 3 DIV and stimulated with 100 µM Ap 5 A in the presence or absence of the P2X1 antagonists NF279 (20 nM), Ip 5 I (10 nM), and NF449 (10 nM). Neurons were stained with an anti-β-III-tubulin antibody to observe their morphology. Scale bar: 25 μm. Quantification of dendritic length ( C ) and the number of dendrites ( D ) from the experiments shown in B . The data represent the mean ± s.e.m. obtained from three independent experiments each involving at least 30 neurons. Statistical differences were analyzed using a one-way ANOVA test followed by Dunnett test correction (* P < 0,05; ** P < 0.01; *** P < 0.0001 versus control)

Article Snippet: The human P2X1 full length cDNA was purchased and sequenced by Geneservice Ltd. (cDNA clone number OHu21348, IMAGE: 5,206,874; Cambridge, UK).

Techniques: Cell Culture, Staining

Journal: Purinergic Signalling

Article Title: Diadenosine pentaphosphate regulates dendrite growth and number in cultured hippocampal neurons

doi: 10.1007/s11302-023-09944-z

Figure Lengend Snippet: P2X1 overexpression reduces the length and number of dendrites but the P2X1R knockdown fail to revert the Ap 5 A-induced reduction of dendrite length. A Western blot analysis of HEK293T cells co-transfected with plasmids overexpressing P2X1-GFP plus different shRNA candidates against P2X1R. The levels of β-actin were used as a loading control. B Representative GFP-fluorescence images of hippocampal neurons transfected at 1 DIV with shLuc-GFP, P2X1-GFP plus shLuc-GFP, or P2X1-GFP plus shRNAP2X1-2 in the presence or absence of 100 µM Ap 5 A. Neurons were fixed, and their dendritic length and ramifications were analyzed at 4 DIV. Quantification of dendritic length ( C ) and the number of dendrites ( D ) from the experiments shown in B . The data represent the mean ± s.e.m. obtained from three independent experiments each involving at least 15 neurons. Scale bar: 25 µm. Statistical differences were analyzed using a one-way ANOVA test followed by Dunnett test correction (* P < 0.05 versus control)

Article Snippet: The human P2X1 full length cDNA was purchased and sequenced by Geneservice Ltd. (cDNA clone number OHu21348, IMAGE: 5,206,874; Cambridge, UK).

Techniques: Over Expression, Western Blot, Transfection, shRNA, Fluorescence