Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: ZIP-ZnT1 complexes mediate a local Zn 2+ -cycle regulating neuronal Zn²⁺ transport

doi: 10.1007/s00018-026-06137-w

Figure Lengend Snippet: ZIP1 and ZIP3 localization in the DCN. A . Immunofluorescent imaging of DCN slices labeled with ZnT3 (red) and the postsynaptic markers GluN2A or CaMKII (green). B . Immunofluorescent staining of ZnT3, ZIP1 or ZIP3 (red) with the synaptic terminal marker VGLUT1 (green). C . GluN2A or CaMKII (green) immunofluorescent co-labeling with ZnT1, ZIP1 or ZIP3 (red), bottom panels show colocalization analyses, represented by Mander’s colocalization coefficient, calculated between the postsynaptic marker and each of the Zn 2+ transporters. For GluN2A colocalization, Tukey’s multiple comparisons test analysis shows a significant difference between ZnT3 and ZIP3 p-value: 0.016, as well as between ZnT3 and ZnT1 p-value: 0.031, F (1.669, 4.451) = 22.32. In case of CaMKII, Tukey’s multiple comparisons test shows significance for ZIP3 p-value: 0.009 and ZnT1 p-value: 0.016, F (3, 15) = 7.202. D . Immunofluorescent staining of PAX6 or Tbr2 (green) with ZIP1 or ZIP3 (red). The colocalization analyses for PAX6, bottom panel, shows Mander’s coefficients of the two Zn 2+ transporters (Mann Whitney test, p-value: 0.003). White arrows indicate examples of cells that show co-localization of the markers

Article Snippet: GluN2A , NeuroMab , 75,288 , 1:300.

Techniques: Imaging, Labeling, Staining, Marker, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: ZIP-ZnT1 complexes mediate a local Zn 2+ -cycle regulating neuronal Zn²⁺ transport

doi: 10.1007/s00018-026-06137-w

Figure Lengend Snippet: ZIP3 and ZnT1 are in direct contact, which enhances ZnT1 efflux rates. A. Co-immunoprecipitation assay in SH-SY5Y cells over expressing ZnT1 and ZIP3, with or without the murine subunit GluN2A (mGluN2A) or the human glutamate receptor (hGluN2A) subunit. The samples were immunoprecipitated with an antibody against ZnT1. The lysates were then separated on SDS-PAGE and subjected to immunoblotting with antibodies against ZIP3, representative of 3 repetitions. Lysis buffer shown as control. B. Co-immunoprecipitation in HEK293 cells expressing ZnT1 and ZIP3. Samples were immunoprecipitated either with an antibody against ZnT1 (left) or ZIP3 (right). Lysates were then separated on SDS-PAGE and subjected to immunoblotting with antibodies against ZIP3 or ZnT1, representative of 3 repetitions. Lysisbuffer shown as control. Note that expected MW for ZIP3 is 37kDa, identified MW likelyrepresents its dimerization and glycosylation; full blots shown in Supplementary Information. C. Representative traces of FluoZin-3 fluorescence changes in SH-SY5Y cells expressing ZnT1+ZIP3 (orange) or ZnT1+PCDNA as control (blue). Cells were perfused with 200μM Zn2+ in Ringer’s solution added with or without pyrithione (see Methods) at the indicated time. The initial Zn 2+ influx and efflux rates were monitored and compared between the two conditions. Unpaired ttest analysis, *** p-value: 0.0003, t(28)=4.131

Article Snippet: GluN2A , NeuroMab , 75,288 , 1:300.

Techniques: Co-Immunoprecipitation Assay, Expressing, Immunoprecipitation, SDS Page, Western Blot, Lysis, Control, Glycoproteomics, Fluorescence

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: ZIP-ZnT1 complexes mediate a local Zn 2+ -cycle regulating neuronal Zn²⁺ transport

doi: 10.1007/s00018-026-06137-w

Figure Lengend Snippet: ZIP3- ZnT1 physical interaction in DCN cartwheel cells. A . Proximity ligation assay (PLA) performed on DCN slices using probes for GluN2A and either ZnT1, ZIP3, or ZIP1. Analysis of PLA puncta (red) was done on the molecular layer (outlined in white) to assess protein–protein interactions in postsynaptic cells of the parallel fibers. The graph (right panel) quantifies PLA puncta normalized to ZnT1-GluN2A puncta. Statistical analysis Dunnett’s multiple comparisons test, p-value: 0.0014, F (2, 11) = 11. B . Co-immunoprecipitation assay using DCN and hippocampal lysates. Protein samples were immunoprecipitated with antibodies against GluN2A, followed by SDS-PAGE separation and immunoblotting with antibodies against ZIP3 to confirm physical interactions. Note that expected MW for ZIP3 is 37 kDa, identified MW likely represents its dimerization and glycosylation; full blots shown in Supplementary Information. C . Schematic representation of Zn 2+ transporters in DCN, indicating the ZIP3-ZnT1-GluN2A complex on the cartwheel cells of the DCN

Article Snippet: GluN2A , NeuroMab , 75,288 , 1:300.

Techniques: Proximity Ligation Assay, Protein-Protein interactions, Co-Immunoprecipitation Assay, Immunoprecipitation, SDS Page, Western Blot, Glycoproteomics

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: ZIP-ZnT1 complexes mediate a local Zn 2+ -cycle regulating neuronal Zn²⁺ transport

doi: 10.1007/s00018-026-06137-w

Figure Lengend Snippet: ZIP1- ZnT1 physical interaction in the hippocampus. A . Co-immunoprecipitation assay using SH-SY5Y cells that were immunoprecipitated with ZnT1 and then exposed to ZIP1 (left panel) or ZIP3 (right panel) antibodies. Note that the right panel is taken from the gel presented in Fig. , the right lane for mGluN2A-ZIP3-ZnT1 expressing cells overlaps with that figure. Note that expected MW for ZIP3 is 37 kDa, identified MW likely represents its dimerization and glycosylation; full blots shown in Supplementary Information. B . Representative traces of fluorescent imaging of FluoZin-3 changes in SH-SY5Y cells expressing ZnT1 + ZIP1 (green) or ZnT1 + PCDNA as control (blue). Cells were perfused with 200 µM Zn 2+ in Ringer’s solution added at the indicated time, with or without pyrithione (see methods). The initial Zn 2+ influx (middle panel) and efflux (right panel) rates are shown in the bar graphs. Unpaired t-test analysis, *** p-value: 0.0001, t(23) = 5.613. C . Co-immunoprecipitation assay using DCN and hippocampal lysates. Protein samples were immunoprecipitated with antibodies against ZIP1, followed by SDS-PAGE separation and immunoblotting with antibodies against GluN2A. D . Proximity ligation assay (PLA) performed on CA3 hippocampal slices using probes for GluN2A and either ZnT1 or ZIP1. Red puncta represent the GluN2A-ZnT1 or ZIP1 interaction in the CA3 pyramidal cell layer (marked by white lines). Analysis of PLA puncta, performed with the GluN2A and either ZnT1, ZIP1 or ZIP3, on CA3 pyramidal layer to assess protein–protein interactions in postsynaptic cells. The graph (bottom panel) quantifies PLA puncta normalized to ZnT1-GluN2A puncta. Statistical analysis Dunnett’s multiple comparisons test, p-value: 0.013, F (2, 6) = 9.81. E . Schematic presentation of the Zn 2+ -cycle proteins, ZIP1-ZnT1-GluN2A, expressed on the postsynaptic CA3 pyramidal cells that are adjacent to the ZnT-3 and ZIP3 expressing mossy fiber terminals, consistent with previous work

Article Snippet: GluN2A , NeuroMab , 75,288 , 1:300.

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Expressing, Glycoproteomics, Imaging, Control, SDS Page, Western Blot, Proximity Ligation Assay, Protein-Protein interactions