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Fig. 1. Plasma membrane expression, oligomeric assembly, and function of affinity- and TagRFP-tagged mP2X2 receptors. a, b For biochemical analysis, X. laevis oocytes expressing the indicated mP2X2 subunit tagged at the N-terminus (N) or C-terminus (C) with a His-(H), StrepII (S), or TagRFP-tag (R) were surface labeled with the membrane-impermeant fluorescent Cy5 dye before protein purification. The indicated proteins were purified under non-denaturing conditions from X. laevis oocytes by Ni2+-NTA chromatography, resolved by BN-PAGE (4–20%) in its non-denatured (native, no SDS) or partially denatured (0.1% SDS) state and visualized by typhoon fluorescence scanning. a Cy5-labeled surface form of the indicated mP2X2 proteins in their native or partial SDS denatured forms as indicated. b TagRFP fluorescence of the BN-PAGE gel shown in a. The number of protomers included in the respective bands is schematically indicated at the right margin. c, d Subcellular distribution and function of mouse P2X2-StrepII-His-RFP receptor construct transiently expressed in HEK293 cells. c Confocal laser scanning microscopy shows plasma membrane bound TagRFR fluorescence, which is also highlighted by the fluorescence intensity diagram along a cross section through the cell. d Representative current trace recorded by the whole-cell patch-clamp technique of a HEK293 cell transiently expressing the mouse P2X2-StrepII-His-RFP receptor construct. The horizontal black bar indicates the duration of application of the agonist 2-meS-ATP (10 µM). Scale bar: c = 10 µm
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Fig. 2. Transgenic P2X2 mice line C57BL/6 J-Tg(RP23-333M22P2X2-StrepHis-TagRFP). a Schematic illustration of a region of the mouse chromosome 5 indicating the position of the origin of BAC clone RP23-333M22 (vertical red line). The BAC clone RP23-333M22 containing the full-length P2RX2 gene (red box) is shown below. The P2RX2 gene is shown with its 11 exons. The StrepII-His-TagRFP encoding sequence (StrepII-His-TagRFP cassette) was inserted directly upstream of the stop codon (exon 11). b–j Confocal laser scanning microscopy of TagRFP fluorescence and TagRFP-/neuronal co-immunostaining. b–d show an overlay of confocal TagRFP fluorescence imaging and transmitted light imaging. Images of a b native dorsal root ganglion, c–g cultured DRG neurons c, d native; e–g PFA fixed), and h–i PFA-fixed cryosections of the dentate gyrus/CA4 region of the hippocampus of an adult C57BL/6 J-Tg(RP23-333M22P2X2-StrepHis-TagRFP) mouse. b Native TagRFP-fluorescence (red) of a crush preparation of a dorsal root ganglion. c, d Native TagRFP-fluorescence (red, arrows) of cultured DRG neurons (2 days in vitro (DIV 2)). e, f TagRFP and MAP2 co-immunostaining (overlay), TagRFP red, MAP2 green; antibodies (ab)) of PFA-fixed cultured DRG neurons (DIV 2). Only a fraction of MAP2-positive DRG neurons do co-express the P2X2-StrepHis-TagRFP transgene (arrows). g TagRFP and MAP2 co-immunostaining (overlay, TagRFP red, MAP2 green) of PFA-fixed cultured DRG neurons of a wild-type C57BL/6 J mouse (littermate control). No TagRFP staining was found. h–j TagRFP and MAP2 co-immunostaining (overlay, TagRFP red, MAP2 green) of PFA-fixed cryosections of the dentate gyrus/CA4 region of the hippocampus. h, i C57BL/6 J-Tg(RP23-333M22P2X2-StrepHis-TagRFP) mouse (adult), j wild-type C57BL/6 J mouse (littermate control). Scale bars: b, c, e, h = 50 µm; d, f, g, i, j = 10 µm
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Fig. 3. CNS Expression of P2X2-TagRFP in transgenic P2X2 mice. Confocal imaging of TagRFP immunofluorescence (red) and Hoechst staining (blue) of PFA-fixed cryosections of the indicated CNS regions of the transgenic P2X2 mice line C57BL/6 J-Tg(RP23-333M22P2X2-StrepHis-TagRFP). TagRFP immunofluorescence and Hoechst staining in coronal sections of the medulla within the brain stem a, b and in fibers within the nucleus tractus solitarius (NTS) c of the cerebellum d–f and g–i and the spinal cord j–l. a, d, j Overlay of Hoechst staining and transmitted light imaging. b, c and e, f and k, l Overlay fluorescence imaging of TagRFP immunofluorescence and Hoechst staining in different magnifications. The arrows in a, b or c show the central canal or TagRFP immunofluorescence in fibers within the NTS, respectively. The arrows in e, f or j–l highlight the plasma membrane bound TagRFP staining in Purkinje neurons or the small fraction of TagRFP staining within superficial dorsal horn laminae of the spinal cord, respectively. g–i immunofluorescence of TagRFP (red) and calbindin (green) and Hoechst staining in the cerebellum. g Overlay of TagRFP immunofluorescence and Hoechst staining. h Overlay of calbindin immunofluorescence and Hoechst staining. i Overlay of calbindin and TagRFP immunofluorescence and Hoechst staining. Scale bars: a, d = 100 μm; b, e, j, k, l = 50 μm; c, f, g, h, i = 20 µm
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Fig. 4. Transgenic P2Y1R mice line C57BL/6 J-Tg(RP23-452G4P2RY1-TagRFP). a Schematic illustration of a region of the mouse chromosome 3 indicating the position of the origin of BAC clone RP23-452G4 (vertical red line); BAC clone RP23-452G4 containing the full-length P2RY1 gene (red box) is shown below. The P2RY1 gene includes two exons. Exon 1 (E1) contains the entire protein-coding sequence of the P2Y1R. The TagRFP-pA encoding sequence (TagRFP-pA cassette) was inserted directly upstream of the P2RY1 translation start site (ATG). b–g cLSM imaging of TagRFP fluorescence and astroglial co-immunostaining. Images of PFA-fixed cryosections of the dentate gyrus (GD)/CA4 region of the hippocampus of an adult C57BL/6 J-Tg(RP23-452G4P2RY1-TagRFP) mouse. b, d, e, g Native TagRFP-fluorescence (red). c, f Immunostaining with the astrocyte marker GFAP (antibody, ab) revealed that GFAP co-localizes with TagRFP, indicating that astrocytes (some are exemplarily marked by thin arrows) express the P2Y1R. Furthermore, also GFAP negative neurons (thick arrow in b, d, e, g) express the P2Y1R within the hippocampus as well. d, g Overlay of native TagRFP fluorescence, GFAP and Hoechst staining. h, i Scratch lesion in a neuron-glia mixed hippocampal cell culture (DIV 10). Four days after lesion outgrowing (or newly formed) TagRFP-positive and GFAP-positive astrocytes were observed in the injury area (arrows). Scale bars: b–j = 20 µm
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Fig. 5. CNS Expression of TagRFP in transgenic P2Y1 reporter mice. Confocal imaging of TagRFP immunofluorescence (red), Hoechst staining (blue) and GFAP, TH or MAP2 co-immunofluorescence (green) as indicated of PFA-fixed cryosections of the indicated CNS regions of the transgenic P2Y1R mice line C57BL/6 J-Tg(RP23-452G4P2RY1-TagRFP). TagRFP immunofluorescence and Hoechst staining in sections of the olfactory bulb a–c, the VTA d–f, the cerebellum g–i, and the spinal cord j–l. a–c show TagRFP immunofluorescence in neurons of the mitral cell (ML) layer and at the border between the external plexiform layer (EPL) and the glomerular layer (GL) of the olfactory bulb, but no co-localization with GFAP co-immunofluorescence a, c. d–f TagRFP immunofluorescence in tyrosine hydroxylase (TH)-positive dopaminergic neurons (arrows) and also a fraction of TH-negative neurons of the VTA. g–i TagRFP immunofluorescence in Purkinje neurons of the cerebellum. GFAP co-immunofluorescence g, i shows no co-localization with TagRFP a,c. j–l TagRFP immunofluorescence in MAP2-positive neurons of laminae I-II, and with less intensity in laminae III-V of the dorsal horn of the spinal cord (Th3). The arrows show neurons with strong MAP2/TagRFP co-staining. Scale bars: c, f, i, l = 50 µm
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Fig. 6. Changes of TagRFP fluorescence during breeding of the transgenic P2Y1R mice line C57BL/6 J-Tg(RP23-452G4P2RY1-TagRFP). Screening of native TagRFP fluorescence in hippocampal cryosections (CA1/2 as indicated) of different generations of the transgenic P2Y1R mice line C57BL/6 J-Tg(RP23-452G4P2RY1-TagRFP). a Representative strong native TagRFP-fluorescence in neurons (cell body, cell processes) of first generations (first year of breeding) of C57BL/6 J-Tg(RP23-452G4P2RY1-TagRFP) mice. b, c Representative native TagRFP-fluorescence in later generations (> 1.5 years of breeding) of C57BL/6 J-Tg(RP23-452G4P2RY1-TagRFP) adult mice. TagRFP fluorescence appears in form of granules in the neuronal cell bodies (arrows). Additionally, native TagRFP-fluorescence-positive processes were still observed. d Finally, after > 2 years of breeding, characteristic cellular native TagRFP-fluorescence disappeared. Only some accumulation of granules in the cell bodies was still found. Scale bars: a, c, d = 20 µm; b = 10 µm
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