Dynasore

Labeling of surface proteins for steady-state cell surface biotinylation were performed as reported previously in cultured cortical neurons (60 (link)). Briefly, biotinylated proteins were precipitated with Pierce NeutrAvidin UltraLink Resin (Thermo Fisher Scientific), and the samples were separated by SDS-PAGE. Surface and total proteins were visualized by Western blotting. To block clathrin-mediated endocytosis, dynasore (Adipogen Life Sciences) and chlorpromazine hydrochloride (Tokyo chemical industry) were used. For the internalization assay, surface proteins were labeled with EZ-Link Sulfo-NHS-SS-Biotin (Thermo Fisher Scientific) for 20 min at 4 °C. Excess biotin was quenched with 25 mM Glycine, then incubated with NH₄Cl at 37 °C for appropriate times. For the 0 min time point, cells were kept at 4 °C as a control. After incubation, cells were quickly washed with ice-cold PBS to stop internalization, and remaining cell surface biotin was cleaved with 50 mM glutathione (Sigma Aldrich) for 30 min at 4 °C. Cells were then extracted in lysis buffer, as described previously (59 (link), 61 (link)). Biotinylated proteins were precipitated with Pierce NeutrAvidin UltraLink Resin, and samples were separated by SDS-PAGE. To detect total surface proteins, we prepared cells labeled with EZ-Link Sulfo-NHS-SS-Biotin without cleavage.

To monitor NSC proliferation, we analyzed NSCs by immunostaining of mouse anti–Ki-67 antibody (BD Pharmingen) and Click-IT EdU detection (Invitrogen) after 4 h exposure to EdU, and co-stained with rabbit anti-Sox2 antibody (EMD Millipore) or DAPI (Sigma) to count all cells. Cell images were obtained on AF6000 (Leica) and BZ-X (Keyence). Cell counting was performed using the ImageJ software for images captured on the AF6000, and Hybrid cell count (Keyence), an algorithm for cell counting, for images captured on the BZ-X. Six to eight images were collected under each condition. Inhibitors used for cell culture were obtained from the indicated suppliers: bafilomycin A1 (Sigma), SAR405 (Cayman Chemical), Dynasore (Adipogen), thapsigargin (Adipogen), nocodazole (Adipogen), DAPT (Calbiochem), AG1478 (Cell Signaling Technology), Torin-1 (Cayman Chemical), and rapamycin (Wako). To measure protein stability, cells were incubated with 10 µg/ml cycloheximide (Sigma) to block protein synthesis.