Mouse cerebral cortical neurons, N2A cells, HeLa S3 cells, and human projection neurons derived from hiPSCs were fixed using 4% PFA for 20 min at room temperature (RT) and washed with phosphate-buffered saline (PBS) + 0.3% Triton X-100. Antigen retrieval was performed for nucleofected cerebral cortical neurons using 10 mM sodium citrate (pH 9) for 1 hour at 60°C. After washing, neurons were incubated in blocking solution (PBS + 0.3% Triton X-100 + 10% normal donkey serum) for 1 hour at RT. Following overnight incubation with primary antibodies (Table 1) in blocking solution at 4°C, washing, incubation with secondary antibodies (PBS + 0.3% Triton X-100 + 1% normal donkey serum) at RT for 1 hour, and washing, coverslips were mounted on a microscope slide using Mowiol.

D. melanogaster larvae were dissected in PBS to expose brain and motor neurons, after dissection larvae were fixed with 4% PFA for 20 min at RT, washed with PBS + 0.2% (CSP staining) or 0.3% (α-tubulin acetylation staining) Triton X-100, and incubated in blocking solution PBS and 0.2% (CSP staining) or 0.3% (α-tubulin acetylation staining) Triton X-100 + 1% bovine serum albumin (BSA) for 30 min at RT. Following overnight incubation with primary antibodies at 4°C, washing, and incubation with secondary antibodies at RT for 2 hours, the larvae were mounted on a microscope slide using Mowiol. After washing in PBS + 0.3% Triton X-100, samples were incubated in blocking solution (PBS + 5% normal donkey serum + 0.3% Triton X-100) for 1 hour at RT. Following overnight incubation with antibodies at 4°C, washing, and incubation with secondary antibodies at RT for 2 hours, the larvae were mounted on a microscope slide using Mowiol. Images were acquired with a Nikon A1Ti confocal microscope (60× lens) for all analyses, except ATAT1-GFP and BDNF-mCherry staining that was acquired with the Airyscan superresolution module of a Zeiss LSM 880 confocal microscope. HeLa cells were plated at densities of 10,000 for 96-well black-bottom plates. The following day, cells were transfected using calcium phosphate with 0.01 μg/96-well plates of ATAT1:IRES-GFP truncated-form plasmids. Twenty-four hours after transfection, cells were fixed using 4% PFA and blocked and permeabilized using PBS + 3% fetal bovine saline (FBS) + 0.1% Triton X-100 for 1 hour. Following overnight incubation with primary [total α/β-tubulin (ATN02-A) and acetylated α-tubulin (T9026)] antibodies at 4°C, washing, and incubation with secondary antibodies at RT for 2 hours, cells were imaged using an IN Cell 2200 microscope (GE Healthcare).

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