Anti ha antibody clone 12ca5

MDCK cells were seeded at 3 x 10⁵ cells per well in a 6-well plate and 24 h later infected with a MOI of 1 of PR8-NS1(1–73)GFP virus or wild type PR8 virus. Mock infected MDCK cells were included as negative control. After 24 h, the cells were lysed on ice for 30 min in 250 μl lysis buffer (50 mM Tris pH 8, 150 mM NaCl, 1% NP40, 5 mM EDTA with protease inhibitors (Complete; Roche Diagnostics N.V. Belgium)). Laemmli buffer containing β-mercaptoethanol was added and the sample was boiled for 10 min. The proteins were separated using SDS-PAGE, and GFP was visualized by western blot, using an anti-GFP antibody (A21311; Molecular probes), a monoclonal anti-HA antibody (clone 12CA5, 11583816001, Roche) or a monoclonal anti-NS1 antibody (sc-130568; Santa Cruz Biotechnology).

All the colorectal cancer cell lines used in this study were maintained in Dulbecco’s modified Eagle’s medium (DMEM; PAA Laboratories) supplemented with 10% fetal bovine serum (PAA Laboratories) and 1x antibiotic-antimycotic (10,000 U penicillin, 10,000 μg streptomycin and 25 μg/ml amphotericin B; Invitrogen) at 37 °C in a humidified atmosphere with 5% CO₂. All cell lines were obtained from the American Type Culture Collection (ATCC) and cell line identity was validated by Affymetrix SNP6.0 array analysis. To stably overexpress EPHB6, LIM2405 and HCT15 cell lines were transfected with a construct expressing N-terminally Hemagglutinin (HA) tagged EPHB6 or the corresponding empty vector control (pDISPLAY Vector, Invitrogen) using Lipofectamine 2000 (Invitrogen). After neomycin selection (500 μg/ml), cells were stained with an anti-HA antibody (clone 12CA5; Roche) and HA-positive cells sorted with a Cell Sorter FACSAria (Becton Dickinson). After expanding the HA-positive population, the sorting was repeated. SW480 and SW620 cells were transduced with a lentiviral vector (pLKO; Sigma) expressing a shRNA with confirmed specificity to EPHB6 (Sequence: CCG GAT GTG GGA AGT GAT GAG TTA TCT CGA GAT AAC TCA TCA CTT CCC ACA TTT TTT; TRCN0000010677, Sigma).