Odessey infrared imaging system

Cells were harvested and lysed in RIPA lysis buffer, and the lysate was subsequently subjected to polyacrylamide gel electrophoresis. The proteins in polyacrylamide gel were then transferred onto a PVDF membrane by the method of wet electrophoretic transfer. The membrane was then blocked in 5% skimmed milk for 1 h and incubated with the primary antibodies at 4 °C overnight. After washing in TBST for 3 times, the membrane was incubated with the secondary antibodies for 1 h at room temperature. Then the levels of proteins were detected by Odessey infrared imaging system (LICOR, Lincoln, Nebraska, USA) or chemiluminescence imaging analysis system (Tanon, Shanghai, China).

Cells were collected with trypsin and pelleted together with non-adherent cells. Cell pellets were washed with PBS and digested with Protein Extraction Reagent Type 4 (MilliporeSigma) with added HALT protease inhibitor cocktail (ThermoFisher), PMSF protease inhibitor (ThermoFisher), and Benzonase nuclease (MilliporeSigma) on ice for 25 min, mixed with loading buffer, and heated at 95 °C for 10 min under reducing conditions. Proteins were separated by SDS-polyacrylamide gel electrophoresis using Any kD TGX stain free gels (BioRad) and transferred to nitrocellulose membranes. Membranes were probed with anti-vinculin mouse monoclonal antibody (Santa Cruz Biotechnology, catalog number sc-73614) and either rabbit polyclonal anti-Zika virus NS4B (GeneTex, catalog number GTX133311) or rabbit polyclonal XBP1 (Invitrogen, catalog number PA5-27650), followed by incubation with secondary antibodies, donkey anti-rabbit IRDye 800CW (LI-COR, catalog number 926-32213), and goat anti-mouse IRDye 680RD (LI-COR, catalog number 926-68070). The blots were imaged with an Odessey Infrared Imaging System (LI-COR Biosciences) and relative density units were calculated with Image Studio Lite version 5.2 and normalized to vinculin.