Isg15

Western blot assay was carried out as follow: cells were lysed with RIPA buffer (Santa Cruz, USA) and cleared lysate was collected by centrifugation for protein separation on 10% SDS-polyacrylamide gel. The proteins were transferred onto PVDF membranes (Millipore) and detected with respective antibodies at 4°C overnight, followed by incubation with either IRDye Fluor 680-labeled IgG or IRDye Fluor 800-labeled IgG secondary antibody (Li-Cor Bioscience). The images were scanned and quantified by densitometric analysis by Li-COR Odyssey Infrared Imager. Primary antibodies against VP1 (Millipore), CD81 (Cell Signaling Technology), IRAK1 (Santa Cruz), CD63 (Abcam), ISG15 (Abcam) and Rab27a, TRAF6, STAT1, TSG101, BST-2/Tetherin, and GAPDH (all from Proteintech), VP2, 3AB, 3C and 3D (all from Genetex) were used.

The following primary antibodies were used in this study: STAT1 (Cell Signaling Technology Inc., #14994), β-actin (Abcam, Inc., #ab115777), TRIM25 (#abs143488), Cathepsin S (#abs137723), NR3C1 (#abs100327), CDKN1B (#abs100463), Arginase 1 (#abs127734), ISG15 (#abs113675), and NOX2 (cat. #abs124860), which were from Absin Bioscience.

Total protein was extracted from the cell lysates using the Western and IP Cell lysis Buffer (Sangon Biotech (Shanghai) Co., Ltd., China) according to the manufacturer’s instructions and protein concentration was determined using the BCA protein assay kit (Sangon Biotech (Shanghai) Co., Ltd., China). After gel electrophoresis, the proteins were transferred to nitrocellulose membranes (Bio-Rad, USA), and blocked in 5% skim milk at 4 °C overnight. After washing with PBST (0.5% Tween-20 in PBS), the membrane was incubated with primary antibodies for 2 h at 37 °C. After washing, the membrane was incubated with horseradish peroxidase (HRP)-conjugated IgG antibody (Abcam, No: ab170487) for 1 h at 37 °C. the protein bands were detected using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Scientific, USA) and chemiluminescence imaging system (BIO-RAD, ChemiDoc MP, California, USA). The primary antibodies of ISG15 (No: ab233071), OAS1(NO: ab86343), Mx1(No: ab95926) and β-actin (No: ab179467) was purchased from Abcam.