For immunoblotting of yeast S. cerevisiae whole-cell lysates, cells were cultured and lysates were prepared as previously described in SDS loading buffer28 (link). Proteins were resolved by SDS-PAGE and blotted using affinity-purified guinea pig anti-Gle1 (ASW 4310 (link)), rabbit anti-GFP (Thermo Fisher Scientific, Waltham, MA), or anti-yPgk1 (Thermo Fisher Scientific). IRDye 800CW-conjugated goat anti-mouse (LI-COR, Lincoln, NE) or Alexa Fluor 700 goat anti-rabbit (Thermo Fisher Scientific) antibodies (1:5000) were visualized with the Li-Cor Odyssey scanner (Lincoln, NE).
For HeLa cell lysate immunoblotting, cells were grown in 60 mm dishes (Fisher Scientific, Pittsburg, PA) and lysed in RIPA buffer (Sigma, St. Louis, MO) supplemented with EDTA-free cOmplete protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). Proteins were resolved by SDS-PAGE and blotted with rat anti-mCherry (Sigma), anti-hNup42/NUPL2 (Sigma), mouse anti-beta-actin (Sigma), or anti-hGle117 (link) antibodies. Infrared 700- or 800-conjugated secondary antibodies (Thermo Fisher Scientific) were visualized with the Li-Cor Odyssey scanner.
For HeLa cell lysate immunoblotting, cells were grown in 60 mm dishes (Fisher Scientific, Pittsburg, PA) and lysed in RIPA buffer (Sigma, St. Louis, MO) supplemented with EDTA-free cOmplete protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). Proteins were resolved by SDS-PAGE and blotted with rat anti-mCherry (Sigma), anti-hNup42/NUPL2 (Sigma), mouse anti-beta-actin (Sigma), or anti-hGle117 (link) antibodies. Infrared 700- or 800-conjugated secondary antibodies (Thermo Fisher Scientific) were visualized with the Li-Cor Odyssey scanner.
