Cell Culture and Antibody Characterization

RPE1 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) and F12 nutrient mix (1:1) supplemented with 10% fetal bovine serum (FBS) (042-30555, FUJIFILM Wako). HEK293 cells were cultured in DMEM containing 10% FBS. These cell lines were obtained from the American Type Culture Collection or the Japanese Collection of Research Bioresources and were regularly tested for Mycoplasma contamination. Antibodies and their suppliers were as follows: anti-Ac-Tub (T6793, Sigma); anti-γ-tubulin (GTU-88, Sigma); anti-Arl13b (17711-1-AP, Proteintech); anti-IFT88 (13967-1-AP, Proteintech); anti-IFT140 (17460-1-AP, Proteintech); anti-GFP [598, Medical and Biological Laboratories (MBL)]; anti-Flag (M2, Sigma or FLA-1, MBL); anti-Myc (9E10, Santa Cruz Biotechnology). Affinity-purified rabbit antibodies against LRRK1, pT1400-LRRK1, pS1790-LRRK1, and NDEL1 were produced according to a previously described method (Hanafusa et al., 2015 (link); Inaba et al., 2016 (link)). A rabbit antibody against pS155-NDEL1 was produced by MBL by injecting rabbits with the synthetic phosphopolypeptide RNAFLEpSELDEKE (where p stands for the phosphorylated residue), coupled to keyhole limpet hemocyanin and affinity purified. BI 2536 and ciliobrevin were purchased from Selleck Chemicals.