Poly l lysine coated coverslips

Immunocytochemistry was performed using standard techniques. Briefly, HEK293 cells were seeded on poly-l-lysine–coated coverslips (Matsunami Glass Ind. Ltd., Osaka, Japan). FLAG-tagged DAN family protein expression plasmids were transfected (1 μg per well) into the cells using Lipofectamine 3000. After transfection (24 hours), the cells were fixed with 4% paraformaldehyde/PBS (Sigma-Aldrich) for 30 min. Next, the cells were washed with PBS three times and incubated in blocking buffer (10% bovine serum albumin/PBS) for 1 hour, followed by incubation in the mouse monoclonal anti–USAG-1 antibody or anti-FLAG antibody (4 ng/ml) (Sigma-Aldrich) in the blocking buffer overnight at 4°C. To visualize the immunoreactivity, the cells were incubated with Cy3-labeled secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA)/PBS (1:400) after being washed three times with PBS. Nuclear staining was performed using 4′,6-diamidino-2-phenylindole (Thermo Fisher Scientific).

Primary cultures of rat cortical neurons were prepared from cerebral cortices of Sprague-Dawley rats at embryonic day 17 [Japan SLC (Shizuoka, Japan)], as described previously [12 (link),13 (link)]. All animal care and experimental protocols were approved by the Animal Experiment Committee of Takasaki University of Health and Welfare (Authorization No. 1733, 1809, 1913, and 2008), and were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Takasaki University of Health and Welfare. The cells were seeded (1.8 × 10⁶ cells) and cultured in poly-l-lysine-coated 6-well plates [AGC Techno Glass (Shizuoka, Japan)] for RT-PCR, and 8 × 10⁵ cells were cultured on 18-mm-diameter poly-l-lysine-coated coverslips [Matsunami Glass Ind., Ltd., (Osaka, Japan)] in 12-well plates (AGC Techno Glass) for immunostaining. The cells were cultured using Neurobasal medium [Thermo Fisher Scientific (Waltham, MA, USA)] containing B27 supplement (Thermo Fisher Scientific), 2 μg/mL gentamicin (Thermo Fisher Scientific), and 0.5 mM glutamine (Thermo Fisher Scientific). Half of the culture medium was replaced with fresh medium every 3 days. Each experiment was performed at 13 days in culture.

Confocal fluorescence imaging was performed using an inverted LSM 710 confocal microscope (Carl Zeiss, Germany) at room temperature (22–25°C). Transfected cells were reseeded onto poly‐L‐lysine‐coated coverslips (Matsunami, Japan). Each coverslip was moved into the chamber for imaging analysis, which was filled with the 140 NaCl solution. Cells were scanned every 2 sec for 60 cycles. Between the 10th and 11th scan, 10 μmol/L rapamycin in the 140 NaCl solution was added to the chamber; the final rapamycin concentration was estimated to be 1 μmol/L. Acquired data were analyzed by using ImageJ software (NIH, USA).