Anti nedd4

Western blotting was performed with anti-myc (Developmental Studies Hybridoma Bank at the University of Iowa, deposited by Dr. J. Michael Bishop, catalog no. 9E 10), anti-HA (Clontech, catalog no. 631207), anti-hIFITM3 (Proteintech Group, catalog no. 11714-1-AP), anti-mIFITM3 (Abcam, catalog no. ab65183), anti-NEDD4 (Millipore, catalog no. 07–049), anti-FLAG (Sigma, catalog no. F7425), anti-actin (Abcam, catalog no. ab3280), or anti-GAPDH (Invitrogen, catalog no. 398600) antibodies. All primary antibodies were used at a 1:1000 dilution. Secondary antibodies, Goat Anti-Mouse IgG, HRP conjugate (Millipore catalog no. 12–349), Goat Anti-Rabbit IgG, HRP-linked (Cell Signaling, catalog no. 70745), and Goat Anti-Mouse, IgG1 Gamma 1 Heavy Chain Specific (SouthernBiotech, catalog no. 1070–05, specifically used for detecting immunoprecipitated protein ubiquitination) were all diluted at 1:20,000.

Cells were harvested 24 h after transfection and lysed for 30 min at 4°C in lysis buffer (50 mM Tris-HCl, pH 7.6, 2 mM EDTA, 150 mM NaCl, 0.5% NP-40) complemented with protease inhibitor cocktail. The cell lysates were immunoprecipitated with anti-FLAG M2 affinity Gel (Sigma-Aldrich) or anti-BST2 (2E6) antibody or the corresponding IgG isotype (Biolegend) coupled to Dynabeads^® Protein G for 4 h at 4°C. Purified proteins were resolved by SDS-PAGE followed by western blotting using anti-HA conjugated to horseradish peroxidase (HRP) (Roche^© Diagnostics), anti-FLAG^®M2 conjugated to HRP (Sigma-Aldrich), anti-GFP conjugated to HRP (Genetex), anti-BST2 (National Institutes of Health) or anti-NEDD4 (Millipore) antibodies.

Anti-NEDD4 was purchased from Millipore (07–049); anti-EGFR (1005) and anti-ACK1 (A11) was from Santa Cruz; anti-PTEN (#9552), anti-AKT (#9272) and anti-phospho-AKT (S473) (#9271) from Cell Signaling; anti-GFP (MMS-118R), anti-HA (MMS-101R) from BioLegend; anti-flag (M2) (F1804) and anti-ACTIN (A5441) from Sigma-Aldrich; anti-EGFR (Mab528) was prepared from culture medium of the EGFR (Mab528) hybridoma cell line (ATCC). The cathepsin B ELISA assay kit was from RayBiotech. Fluorescent dye-conjugated secondary antibodies and phalloidin were purchased from ThermoFisher. The cathepsin B inhibitor CA-074Me was purchased from Apexbio. The NEDD4 shRNA (5’-AUUUGAACCGUAUAGUUCAGC-3′) in the lentiviral expression vector pLKO.1 was purchased from Open Biosystems (RHS4533-EG4734). The lung cancer cell lines A549 and H1650 were purchased from ATCC.