*2.3. Immunofluorescence Assay (IFA)*

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).
