Mouse anti ha

All cell-based assays in this study were carried out in 293T cells. 293T cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Sangon Biotech). Construct transfection was performed using PEI (Sigma) according to the manufacturer’s instructions [54 (link)]. Two days after transfection, cells were harvested to extract total protein for following co-IP and immunoblot (IB) according to our previous protocols [55 (link)]. The following antibodies were used for IP and IB: mouse anti-Flag (1:500 for IP, 1:5000 for IB, Sigma); mouse anti-Myc (1:200 for IP, 1:2000 for IB, Santa Cruz); mouse anti-HA (1:200 for IB); mouse anti-Actin (1:5000, Genscript); rabbit anti-Ub-K63 (1:1000 for IB, ABclonal); rabbit anti-Ub-K48 (1:1000 for IB, ABclonal); goat anti-mouse HRP (1:10000, Abmax) and goat anti-rabbit HRP (1:10,000, Abmax). The densities of IB bands were measured by Image J software.

For the immunofluorescence assay, U2OS cells were grown in 6-well tissue culture plates and transfected with 2 μg of opt-36αt, opt-36βt, or opt-36γt. Two days after transfection, the cells were fixed with 4% paraformaldehyde for 15 min. Nonspecific binding was then blocked with normal goat serum diluted in PBS at room temperature for 1 h. The plates were then washed in PBS for 5 min and subsequently incubated with anti-HA antibody at a 1:1000 (mouse anti-HA, GenScript) dilution overnight at 4 °C. The plates were washed as described above and incubated with appropriate secondary antibody (goat anti-mouse IgG-AF488, Sigma, St. Louis, MO, USA) at 1:200 dilutions at room temperature for 1 h. After washing, DAPI (Millipore Sigma) was added to stain the nuclei of all cells following manufacturer’s protocol. Wells were washed and maintained in PBS, and observed under a microscope (EVOS Cell Imaging Systems; Life Technologies, Carlsbad, CA, USA).