Dylight coupled secondary antibodies

Cells were lysed in 1% Triton‐X/PBS in the presence of protease inhibitor cocktail (cOmplete, Roche) and clarified by centrifugation. Virions were purified from supernatants by overlaying on a 6% OptiPrep cushion and pelleted by ultracentrifugation (50,000 × g, 4°C) for 1 h. An aliquot was removed for p24 Gag ELISA, and normalized amounts were loaded on a Bis–Tris 12% acrylamide SDS–PAGE gel. The following antibodies were used: mouse anti‐FLAG‐M2 (Sigma), mouse anti‐IFITM3 (Proteintech, 66081‐1‐Ig), rabbit anti‐IFITM3 (Abcam, EPR5242), mouse anti‐Gag p24 (183‐H12‐5C, NIH #1513), sheep anti‐Env gp120 (NIH #288), mouse anti‐HA (Covance, HA.11 clone 16B12), rabbit anti‐actin (Santa Cruz Biotechnology), mouse anti‐tubulin (Santa Cruz Biotechnology) and mouse anti‐ubiquitin antibody (FK2, Enzo Life Sciences, recognizing K²⁹‐, K⁴⁸‐, and K⁶³‐linked mono‐ and polyubiquitinated proteins). DyLight‐coupled secondary antibodies (Thermo Fisher) were used for protein detection on a Li‐Cor Odyssey imaging system.

All cell pellets were lysed in a hypotonic lysis buffer (10 mm Tris, pH 7.9, 1.5 mm MgCl₂, 10 mm KCl, 0.5 mm dithiotreitol, supplemented with protease inhibitors from Roche) on ice for 15 min, followed by centrifugation at 2500 × g at 4 °C. The supernatant was saved as the cytoplasmic fraction, and the pellet resuspended in 20 mm HEPES pH 7.5, 1 m NaCl, 20% glycerol, 1.5 mm MgCl₂, 0.1 mm EDTA, 0.5 mm dithiotreitol, and protease inhibitors, then sonicated with a Branson sonicator for 3 s at setting 3. Lysates were spun again at 16,000 × g for 20 min at 4 °C, and the supernatant containing nuclear and chromatin fractions was pooled with the cytoplasmic fraction. Protein concentration was determined via Bradford assay, and equal total protein loads were run on 4–12% gradient Bis-Tris gel (Life Technologies; Grand Island, NY) using MES running buffer, before transferring to 0.2 micron nitrocellulose (Bio-Rad; Hercules, CA). Blots were blocked in 5% milk in PBS, probed with indicated primary antibodies overnight at 4 °C, followed by incubation with DyLight coupled secondary antibodies (Thermo) for 1 h at room temperature, before detection on the LiCor Odyssey imaging system.

Cells were lysed in 1% Triton X‐100/PBS in the presence of protease inhibitor cocktail, and 10–20 μg of protein lysate was loaded into 12% Bis‐Tris SDS–PAGE gels. The following antibodies were used: IFITM1 (Proteintech, 60074‐1‐Ig), IFITM2 (Proteintech, 66137‐1‐Ig), IFITM3 (Abcam, EPR5242/ab109429), actin (Santa Cruz Biotechnology), tubulin (Santa Cruz Biotechnology), and LC3 (MBL International PM036). DyLight‐coupled secondary antibodies (Thermo Fisher) were used for protein detection on a Li‐Cor Odyssey imaging system.