Epr5071

HeLa TREx Flp-In cells were generated from HeLa TREx cells (Thermo catalog no. R71407) using the pFRT/lacZeo vector (Thermo catalog no. V601520) according to the vendor’s protocols. Kata-WT and kata-D210C cell lines were generated from the HeLa TREx Flp-In cells using pOG44 plasmid and the pcDNA5.0/FRT/TO vector containing EGFP-tagged full-length katanin-WT or -D210C (described in Plasmids section of Methods ). Genomic DNA was extracted from cells using the DNeasy Blood and Tissue kit (Qiagen); insertions were PCR amplified and sequenced. Cells were cultured in DMEM (High glucose with sodium pyruvate and L-glutamine, ThermoFisher) supplemented with 10% (v/v) FBS (Sigma-Aldrich) and hygromycin B (200 μg mL⁻¹) at 37 °C and 5% CO₂. Cells were confirmed to be mycoplasma free using a PCR-based method^{43 (link)}.
For western blotting, cells were cultured with doxycycline (10 ng mL⁻¹ for 24 h) before lysis at 4 °C. The following antibodies were used: rabbit monoclonal anti-p60 katanin (1:1000; EPR5071; Abcam) and mouse monoclonal anti-GAPDH (1:1000; 1E6D9; Proteintech). Membranes were imaged using a LI-COR Odyssey Infrared Imager. As previously^{13 (link)}, Cell viability assays were conducted using CellTiter-Glo Luminescent Cell Viability Assay (Promega) according to the manufacturer recommendations. The luminescence signal was quantified using a Synergy Neo Microplate Reader.