Fig 1: Expression and function of A2AA3Hets in primary cultures of microglia from C57BL/6J mice. (A) A2AA3Hets were detected in primary cultures of microglia by the in situ proximity ligation assay (PLA) using specific antibodies. Cell nuclei were stained with Hoechst (blue). Samples from 5 different animals were processed and analyzed. Scale bar: 20 µm. (B–D) Primary cultures of microglia from C57BL/6J mice were pre-treated with antagonists, 1 µM SCH 58261 -for A2AR- or 1 µM PSB 10 -for A3R-, subsequently stimulated with selective agonists, 100 nM PSB 777 -for A2AR- or 100 nM 2-Cl-IB-MECA -for A3R-, individually or in combination and treated with 500 nM forskolin (FK) or vehicle. (B) ERK1/2 phosphorylation was analyzed using an AlphaScreen® SureFire® kit (PerkinElmer) while cAMP levels were collected by the Lance Ultra cAMP kit and results were expressed in % respect to basal levels (B,C) or in % respect levels obtained upon 0.5 µM FK stimulation (D). Values are the mean ± S.E.M. of 10 different experiments performed in triplicates. One-way ANOVA followed by Bonferroni’s multiple comparison post-hoc tests were used for statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; versus basal (B,C) or versus FK treatment (D).
Fig 2: Expression and function of A2AA3Hets in primary cultures of microglia from the APPSw,Ind mice model of Alzheimer’s disease. (A) A2AA3Hets were detected in primary cultures of APPSw,Ind microglia by the in situ proximity ligation assay (PLA) using specific antibodies. The negative control was obtained by omitting the primary anti-A3R antibody. Experiments were performed in samples from 12 different animals. (B) The number of red dots/cell was quantified using the Andy’s algorithm Fiji’s plug-in and represented versus the number of Hoechst-stained cell nuclei (blue). The number of red dots/cell was compared to those in microglia from wild type (WT) mice. The unpaired t-test was used for statistical analysis. ** p < 0.01, versus WT. Scale bar: 20 μm. (C–E) Primary cultures of microglia from APPSw,Ind mice were pre-treated with antagonists, 1 μM SCH 58261 -for A2AR- or 1 μM PSB 10 -for A3R-, and subsequently stimulated with selective agonists, 100 nM PSB 777 -for A2AR- or 100 nM 2-Cl-IB-MECA -for A3R-, individually or in combination. (C) ERK1/2 phosphorylation was analyzed using an AlphaScreen® SureFire® kit (PerkinElmer) while cAMP levels were collected by the Lance Ultra cAMP kit and results were expressed in % respect to basal levels (C,D) or in % respect levels obtained upon 0.5 μM FK stimulation (E). Values are the mean ± S.E.M. of 6 different experiments performed in triplicates. One-way ANOVA followed by Bonferroni’s multiple comparison post-hoc tests were used for statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; versus basal (C,D) or versus FK treatment (E).
Fig 3: Expression of A2AA3Hets in primary cultures of neurons isolated from the fetuses of pregnant C57BL/6J female mice. (A) A2AA3Hets were detected by the in situ proximity ligation assay (PLA) in primary cultures of striatal, hippocampal, and cortical neurons isolated from mouse fetuses. The negative control was obtained by omitting the primary antibody anti-A3R. Experiments were performed in samples from 6 different animals. (B) The number of red dots/cell was quantified using the Andy’s algorithm Fiji’s plug-in and represented versus the number of Hoechst-stained cell nuclei (blue). The number of red dots/cell was compared to those in neurons from different brain regions. The unpaired t-test was used for statistical analysis. * p < 0.05, *** p < 0.001. Scale bar: 10 µm.
Fig 4: A2AA3Het-mediated Gs/Gi signaling in primary striatal, hippocampal, and cortical neurons from C57BL/6J mice. (A–F) Primary neurons were pre-treated with selective antagonists, 1 µM SCH 58261 -A2AR- or 1 µM PSB 10 -A3R-, and subsequently treated with the selective agonists, 100 nM PSB 777 -A2AR- or 100 nM 2-Cl-IB-MECA -A3R-. cAMP levels after 500 nM forskolin (FK) stimulation or vehicle treatment were detected by the Lance Ultra cAMP kit and results were expressed in % respect to basal levels (A–C) or in % respect to levels obtained upon FK stimulation (D–F). The values are the mean ± S.E.M. of 6 different experiments performed in triplicates. One-way ANOVA followed by Bonferroni’s multiple comparison post-hoc test were used for statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; versus basal levels (A–C) or versus FK treatment (D–F).
Fig 5: Expression and function of A2AA3Hets in primary cultures of LPS-activated microglia from C57BL/6J mice. (A) A2AA3Hets were detected in primary cultures of non-activated and LPS-activated microglia by the in situ proximity ligation assay (PLA) using specific antibodies. The negative control was obtained by omitting the primary anti-A3R antibody. Experiments were performed in samples from 5 different animals. (B) The number of red dots/cell was quantified using the Andy’s algorithm Fiji’s plug-in and represented versus the number of Hoechst-stained cell nuclei (blue). The number of red dots/cell was compared between non-activated and LPS-activated microglia. The unpaired t-test was used for statistical analysis. Scale bar: 20 µm. (C–E) Primary cultures of LPS-activated microglia from C57BL/6J mice were pre-treated with antagonists, 1 µM SCH 58261 -for A2AR- or 1 µM PSB 10 -for A3R-, subsequently stimulated with selective agonists, 100 nM PSB 777 -for A2AR- or 100 nM 2-Cl-IB-MECA -for A3R-, individually or in combination and treated with 500 nM forskolin (FK) or vehicle. (C) ERK1/2 phosphorylation was analyzed using an AlphaScreen® SureFire® kit (PerkinElmer) while cAMP levels were collected by the Lance Ultra cAMP kit and results were expressed in % respect to basal levels (C,D) or in % respect to levels obtained upon 0.5 µM FK stimulation (E). Values are the mean ± S.E.M. of 6 different experiments performed in triplicates. One-way ANOVA followed by Bonferroni’s multiple comparison post-hoc tests were used for statistical analysis. * p < 0.05, **** p < 0.0001; versus basal (C,D) or versus FK treatment (E).
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