Anti ifitm3

Antibodies used were as follows: anti-Paxillin (RRID:AB_647289), anti-GM130 (RRID:AB_398141), anti-p130Cas (RRID:AB_397667) and anti-RACK1 (RRID:AB_397577) antibodies (BD Transduction Laboratories, New Jersey, USA), anti-IFITM3 (RRID:AB_2122095) (Abcam, Cambridge, UK), anti-CoxIV (RRID:AB_10694213), anti-FAK (RRID:AB_10694068), anti-pFAK Y397 (RRID:AB_2173659), anti-pPaxillin Y118 (RRID:AB_2174466), anti-Rab7 (RRID:AB_1904103), anti-pSrc Y416 (RRID:AB_331697), anti-Src (clone 36D10) (RRID:AB_10693939), anti-LC3B (RRID:AB_2137707) and anti-GAPDH (RRID:AB_10622025) (Cell Signaling Technologies, Danvers, MA, USA), and anti-Dctn1 (RRID:AB_10842517), anti-Ambra1 (RRID:AB_2636939) and anti-pSrc Y416 (RRID:AB_309898) antibodies and anti-FAK (clone 4.47)-conjugated agarose antibody (RRID:AB_310789) (Millipore, Billerica, MA, USA). LC3B antibody was from MBL (RRID:AB_1279144) (MBL International, Woburn, MA, USA). TRITC-phalloidin was purchased from Life Technologies (RRID:AB_2572408) (Paisley, UK). Anti-rabbit (RRID:AB_2099233) or mouse (RRID:AB_330924) peroxidase-conjugated secondary antibodies were purchased from Cell Signaling Technologies. Dasatinib was obtained from Bristol Myers Squibb (Princeton, NJ, USA) and PF562271 from Pfizer (Groton, CT, USA).

The bEnd.3 cell lysates were prepared by using radioimmune precipitation assay (RIPA) lysis buffer containing complete mini-protease-inhibitor and phosphatase inhibitor cocktail tablets (Roche Diagnostics Corporation, Indianapolis, IN) and centrifuged at 12,000 x g for 10 min at 4°C for collecting supernatants. After determining protein concentrations, the proteins were electrophoresed under reducing conditions in 10% SDS–PAGE gels and transferred to PVDF membranes. The membranes were incubated overnight at 4°C with anti-Claudin-5 (Cat# 35–2500, 1:1000 dilution, Thermo Fisher Scientific, Waltham, MA), anti-STAT1 (Cat#14994S, 1:1000 dilution, Cell Signaling Technology, Danvers), anti-STAT2 (Cat#72604S, 1:1000 dilution, Cell Signaling Technology, Danvers), anti-IFITM3 (Cat#ab15592, 1:1000 dilution, Abcam, Cambridge, MA), and anti-GAPDH antibody (Cat#MAB374, 1:2000 dilution, Sigma-Aldrich, St. Louis, MO). Specific bands were visualized by an enhanced chemiluminescence system (Amersham, Arlington Heights, IL). The optical densities of the protein bands were determined by densitometric scanning using an HP Scanjet G3010 Photo Scanner and Image J V1.40 (NIH, MD). GAPDH was used to normalize expression levels of genes of interesting.