Fig. 1.

Characterization of FLAG-tagged HAs. (A) The FLAG epitope (DYKDDDDK) was inserted in frame into PR/8 (H1N1) HA at site 135, 291, 298, 313, or 366 (residues indicated in red). The receptor-binding region represented by amino acids Y108, W166, H196, and L207 (indicated in green) resides on top of the globular head of the HA. (B) Cell surface expression of FLAG-tagged HAs was assessed via FACS analysis on transfected 293T cells using polyclonal sera, head-specific mAb PY102, an anti-FLAG mAb, or an IgG2a control (mAb XY102). Percent binding for each HA construct was normalized to polylconal sera against each respective HA. (C) Expression of the FLAG epitope of the tagged HAs was further confirmed by Western blot analysis of whole cell lysates from transfected 293T cells using mAb PY102 and an anti-FLAG mAb. GAPDH served as a loading control. (D) Binding of FLAG-tagged HAs to sialic acid motifs was assessed by a modified hemolysis assay. FLAG-tagged HA transfected cells were coincubated with a 0.5% solution of chicken RBCs for 1 h at 4 °C. Heme released from agglutinated transfected cells was quantified by absorbance (405 nm). Error bars represent SEM.