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. 2021 Apr 26;22(9):4511. doi: 10.3390/ijms22094511

Figure 2.

Figure 2

Zip4 is expressed at synapses in neurons and enriched in Purkinje cells of the cerebellum. (A) Expression of the Zip4 gene was detected in brain lysate of wild-type mice (n = 3 animals) and normalized against hmbs. Lowest mRNA levels were detected in cortex and significantly higher levels compared to cortex were found in cerebellum (ANOVA: F = 5.554; p = 0.006; posttest: ctx vs. cer p = 0.0144). (B) Expression of ZIP4 protein was similarly found in all analyzed brain regions (ctx, hip, str and cer), with highest levels observed in cerebellum (ANOVA: F = 15.440; p = 0.003; posttest: ctx vs. cer p = 0.017; hip vs. cer p = 0.006; str vs. cer p = 0.013) (n = 3 animals). (C) High ZIP4 expression was detected in Purkinje cells of the cerebellum. Images show representative stainings of mouse brain sections using ZIP4 antibody and calbindin as a marker for Purkinje neurons (arrows) (scale bar = 250 μm (upper panel) and = 30 μm (lower panel)). (D) ZIP4 expression is highest in the synapse associated protein fraction (P2), but ZIP4 is also found in the fraction containing soluble cytoplasmic proteins (cerebellum: ANOVA: F = 9.737, p = 0.0049, posttest: Ho vs. P2 p = 0.0354, S2 vs. P2 p = 0.0031; Striatum: Ho vs. P2 p = 0.0417, S2 vs. P2 p = 0.0468) (n = 3 per brain region and fraction). (E) Staining of primary hippocampal cultures at DIV14 shows that ZIP4 expression is found in neurons. MAP2 was used to visualize neuronal cells. DAPI was used to visualize cell nuclei. ICC confirms ZIP4 immunoreactive signals at synapses (arrows, zoomed region indicated by rectangle). There, ZIP4 signals colocalize with the postsynaptic marker protein HOMER1b/c (upper panel) but are juxtaposed to signals from the presynaptic marker protein BASSOON (lower panel) (scale bar = 50 μm (upper panels) and = 10 μm (lower panels)). (F) Co-IP experiments confirm that ZIP4 can be found in a complex centered around the postsynaptic scaffold protein SHANK3. SHANK3 was coupled to magnetic beads via SHANK3 antibody. Brain lysate from mice was used, and ZIP4 signal was detected in the input and in precipitated proteins in the eluate. Empty beads did not precipitate ZIP4. (G) Similar to colocalization with HOMER1b/c, ZIP4 signals colocalize with SHANK3 signals in hippocampal neurons in vitro (scale bar = 15 μm).