Nbp1 06712

For acetyl-tubulin immunostaining, cells were seeded on cover glasses at a density of 1 × 10⁵ cells per 12-well plate. The transfected cells were treated with DMSO, 0.8 μg/ml Taxol (Sigma Aldrich #T1912) or 1 μg/ml Colchicine (Sigma Aldrich #C9754) for 6 h. After treatment, the cells were kept in DMEM/F12 for 24 h in 37 °C and then fixed with 4% paraformaldehyde (Electron Microscopy Sciences #15710) in PBS for 10 min at room temperature. Fixed cells were incubated in 0.1% Triton-X (0.1% PBST) 10 min for cellular permeabilization and blocked with 1% BSA in PBS for 1 h in room temperature. After blocking, the cells were stained with anti-alpha-tubulin (1:500) (Abcam #ab6160), anti-acetyl-tubulin (1:500) (Sigma Aldrich #T6793) and anti-FLAG tag (1:500) (Novus Biologicals #NBP1-06712) diluted in the blocking buffer overnight in 4 °C. On the next day, the primary antibodies were removed and cells washed for 5 min three times with 0.4% Tween-20 (0.4% PBST) and incubated in secondary antibody for 1 h at room temperature. The stained cells were mounted in the VECTASHIELD (Vector Laboratories #H-1200) with DAPI for further imaging. The catalog number and the working concentration of both primary and secondary antibodies are described in Appendix Table S7.

HeLa microsomes were prepared according to [49 (link)] and immunoblotting was performed as previously described [113 (link)]. For total cell lysate investigation, cells were lysed using RIPA buffer (ThermoFisher Scientific, 89900) and DNA excluded by centrifugation (15,000×g for 15 min). Briefly, western blots of typically 20–40 µg of protein were ran on 4–12% Bis/Tris gel (NuPage, Thermo Scientific, NP0323BOX) and transferred to a 0.45 µm PVDF membrane (Immobilon-P, Thermo Scientific, 88518) and probed for ATP10B (Sigma-Aldrich, HPA034574), CDC50A (anti-FLAG antibody; Sigma-Aldrich, F3165). Cell death was assessed using a cleaved caspase 3 antibody (Cell signaling, 9661). All blots were probed for either GAPDH (Sigma-Aldrich, G8795) or β-actin (Sigma-Aldrich, C6198) as a loading control. Detection was performed using HRP-conjugated secondary antibodies (BIOKE, 7074S and 7076S). Detections were performed on a Bio-Rad Chemidoc Imager with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, 32106). For co-immunoprecipitation experiments, PVDF membranes (Hybond P; Amersham Biosciences) were blocked in 5% skimmed milk in PBS and probed with primary antibodies against HA-tag (Covance, MMS-101P) and FLAG-tag (Novus Biologicals, NBP1-06712).