*2.2. Western Blot*

Transfections were performed using the TurboFectin 8.0 reagent, following the manufacturer's protocols (OriGene, Rockville, MD, USA). Briefly, U2OS cells were grown to 80% confluence in 6-well tissue culture plates and transfected with 2 μg of opt-36αt, opt-36βt, or opt-36γt. The cells were collected 2 days after transfection, washed twice with PBS and lysed with cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA). Gradient (4–12%) Bis-Tris NuPAGE gels (Life Technologies, Carlsbad, CA, USA) were loaded with transfected cell lysates and transferred to polyvinylidene difluoride (PDVF) membrane. The membranes were blocked in PBS Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE, USA) for 1 h at room temperature. To detect plasmid expression, the anti-HA (A01244 Clone 5E11D8, GenScript, Piscataway, NJ, USA) antibody was diluted 1:1000 and anti–β-actin antibody diluted 1:5000 in Odyssey blocking buffer with 0.2% Tween 20 (Bio-Rad, Hercules, CA, USA) and incubated with the membranes overnight at 4 ◦C. The membranes were washed with PBST and then incubated with the appropriate secondary antibody (goat anti-mouse IRDye680CW; LI-COR Biosciences) at a 1:15,000 dilution in Odyssey Blocking Buffer for 1 h at room temperature. After washing, the membranes were imaged on the Odyssey infrared imager (LI-COR Biosciences).
