Western Blotting Procedure with Antibody Detection

For western blotting, samples were sonicated for 5 min and boiled in a solution of 2× Laemmli Sample Buffer and 10% β-mercaptoethanol before being fractionated on a 4–12% NuPAGE Bis-Tris gel (Invitrogen). The iBlot Gel Transfer system (Invitrogen) was then used to transfer samples to a nitrocellulose membrane. The following antibodies were used at 1:3000–10,000 dilutions: α-Tubulin: α-Tubulin (DM1a; Sigma), α-GFP: IgG₁κ (Roche), α-SAS-5 (Song et al. 2011 (link)), and α-TBG-1 (Stubenvoll et al. 2016 (link)). IRDye secondary antibodies (LI-COR Biosciences, Lincoln, NE) were used at a 1:10,000 dilution. Blots were imaged using the Odyssey infrared scanner (LI-COR Biosciences), and analyzed using Image Studio software (LI-COR Biosciences).