Znt 3

As for DAB staining, mice were transcardially perfused with 4% PFA and postfixed for an additional 24 h. Following immersed into 4% agarose, brains were sectioned into 30 μm thick sagittal sections using the VT1000S vibratome (Leica Microsystems, Wetzlar, Germany). Tissue sections were incubated with 0.1% Triton/PBS and 1 M glycine, followed by blocking with 1% BSA in PBS for 45 min. The sections were then incubated with the primary antibody AT8 (1:100, Invitrogen, CA, USA) overnight. This was followed by washing with PBS and a second incubation for 2 h at room temperature with a second primary antibody against NeuN (1:400, Millipore, Arklow, Ireland), GFAP (1:400, Sigma-Aldrich, Arklow, Ireland), Iba-1 (1:400, Wako, Fuggerstrasse, Germany) or Zinc Transporter 3 (ZnT3; 1:200, Synaptic Systems GmbH, Goettingen, Germany). After washing in PBS, tissue was incubated with fluorescent secondary antibodies [AlexaFluor-488 or AlexaFluor-568 (BioSciences, Dublin, Ireland)] followed by a short incubation with DAPI. FluorSave^TM (Millipore, Arklow, Ireland) was used to cover the tissue, and confocal images were taken with a TCR 6500 microscope (Leica Microsystems, Wetzlar, Germany) equipped with four laser lines (405, 488, 561, and 653 nm) using a 40× immersion oil objective (NA 1.3; Leica Microsystems).

Preparation of the tissue protein was according to the conventional method. Tissues were taken out from mouse brains (midbrain or striatum) or kidneys, and homogenized in RIPA Buffer (#89900, Thermo Scientific) containing protease inhibitor cocktails (P8340, Sigma-Aldrich) and phosphatase inhibitor cocktails (#78420, Thermo Scientific) on ice. The homogenates were centrifuged at 14,000 rpm for 30 min at 4 °C after 30-min incubation on ice. The supernatants were collected and subjected to conventional SDS-PAGE and Western Blotting analyses. SuperSep Phos-tag gels were used for the detection of phospho-AP3B1 (Wako Pure Chemical Industries). The antibodies used here were: Actin (ab3280, Abcam), AP3B1 (ARP33647_P050, Aviva Systems Biology), LIMP2/SCARB2 (PA5-20540, Thermo Scientific), and ZnT3 (#197003, Synaptic Systems).