Expression and purification of soluble H5-HA proteins HEK293T cells stably expressing soluble HA (sHA) proteins were generated by selection of transfected cells in culture medium containing 300 ug/ml hygromycin for at least 1 month. To purify sHA proteins, cells were grown in DMEM with 5% FBS until 90% confluence. Culture supernatant containing secreted sHA proteins was cleared by centrifugation at 4000 rpm for 15 minutes at 4uC, concentrated by Amicon Ultra-15 Centrifugal Filter Units with Ultracel-100 membrane (Millipore, Billerica, MA, USA; 100 kDa cut-off) and stored at 280uC until use. Soluble HA proteins were affinity purified from concentrated supernatant using anti-FLAG M2 affinity gel (Sigma). Because it has been shown that HA-bound sialic acid could interfere with the accessibility of the receptorbinding site to cellular receptors [42] , anti-FLAG M2 resins bound with sHA proteins were washed twice in cold PBS and subjected to treatment with NAvb (Roche, 62.5 mU/ml) at 37uC for 45 minutes to remove terminal sialic acid residues, followed by three washes in cold PBS. Bound sHA proteins were eluted with 150 ml FLAG peptide (International Laboratory, USA, 0.4 mg/ml in PBS) for four times. To remove the FLAG peptides, all eluates were pooled and concentrated using Amicon Ultra-0.5 mL with Ultracel-100 membrane (Millipore, 100 kDa cut-off). To examine the oligomeric state of sHAs, proteins were resolved on a discontinuous native PAGE (6% of acrylamide) followed by western blot detection using a HRP-conjugated anti-FLAG M2 antibody (Sigma).