Cells fixed with 4% paraformaldehyde in PBS, fixed mouse brain samples, or postmortem human temporal lobe sections were blocked with a solution containing 5% normal donkey serum (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), 2% BSA (Sigma-Aldrich), and 0.1% Triton X-100 (Sigma-Aldrich) for 1 hour at room temperature. Samples were then incubated with combinations of primary antibodies against RNF146, pAkt1, TH, or PAR, depending on the experiment, at 4°C overnight. Then, the fixed samples were washed with PBS containing 0.1% Triton X-100 and incubated with corresponding secondary antibodies conjugated with fluorescent dye at room temperature for 1 hour. Fluorescent images were obtained using a fluorescence microscope (Axiovert 200M, Zeiss, Oberkochen, Germany) or confocal microscope (LSM 710, Zeiss).

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