Anti arl13b 17711 1 ap

The mouse mAb anti-SMO clone E5 (sc-166685) was from Santa Cruz (Santa Cruz, USA). The mouse mAb anti-FLAG (clone M2, F3165) was from Sigma (St Louis, USA). The rabbit pAb anti-ARL13B (17711-1-AP) was from proteintech (Rosemont, USA). The Alexa Fluor 488 goat anti-mouse secondary antibody (A11001) and Alexa Fluor 555 donkey anti-rabbit secondary antibody (A31572) were from Thermo Scientific (Waltham, USA). The donkey anti-mouse HRP-conjugated secondary antibody (715-035-150) was from Jackson ImmunoResearch (West Grove, USA). CuSO
₄ (C8027), Vc (A7631) and TBTA (678937) were from Sigma. Cholesterol probe (CP) and biotin alkyne were synthesized by Shanghai Bio Bond Pharmaceutical Co., Ltd (Shanghai, China)
[21] (link).

HUVECs grown on eight‐well cell culture slides (Mat‐Tek) coated with Fibronectin (Corning; Sigma‐Aldrich) were washed with warmed PBS (37°C) twice, fixed in warm 4% paraformaldehyde (Thomas Scientific; 37°C) for 5 min and washed with PBS three times. The cells were permeabilized with 1% Triton X‐100 in PBS for 5 min and washed again with PBS three times. The cells were blocked with 1% bovine serum albumin (Sigma‐Aldrich) in PBS for 30 min. Anti‐acetylated‐tubulin (T6793; Sigma‐Aldrich; 1:1000), anti‐Arl13B (17711‐1‐AP; Proteintech; 1:500), anti‐IFT81 (11744‐1‐AP; Proteintech; 1:500), and anti‐BMPR‐II (612292; BD Biosciences; 1:500) antibodies were incubated overnight at 4°C, and then detected with Alexa Fluor 568 donkey anti‐mouse IgG (A10037; Invitrogen; 1:500), Alexa Fluor 568 goat anti‐rabbit IgG (A11011; Invitrogen; 1:200), Alexa Fluor 488 goat anti‐mouse IgG (A11001; Invitrogen; 1:200), and/or Alexa Fluor 647 goat anti‐rabbit IgG (A21244; Invitrogen; 1:200). 0.5 µg/ml DAPI (Sigma‐Aldrich) was used to stain the nuclei. The cells were mounted using Vectashield (Vector Labs) and imaged using fluorescence microscopy.

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.