Fig 3.

Identification of the VP1u region responsible for binding and internalization. (A) A linear schematic representation of the recombinant full-length VP1u (WT) and the truncated proteins (ΔN29, ΔC128, and ΔN29/ΔC128) is shown. Important regions and epitopes are indicated with different colors: red, the truncated N-terminal region; green, epitope recognized by the neutralizing N-VP1u MAb; blue, epitope recognized by the PLA₂ Ab; violet, FLAG tag used for detection; brown, MAT (His tag) used for purification. The inserted cysteine for protein dimerization is shown as a sulfhydryl side chain. Antibodies used are schematically shown above their corresponding epitopes (N-VP1u Ab, PLA₂ Ab, and anti-FLAG Ab). (B) Internalization assay with full-length (WT) and truncated versions (ΔN29, ΔC128, and ΔN29/ΔC128) of VP1u. Recombinant VP1u proteins were incubated with UT7/Epo cells in the presence of anti-FLAG antibody for 30 min at 37°C. VP1u-mediated uptake of anti-FLAG antibody was detected postfixation by IF with a secondary Alexa Fluor 488-labeled Ab. (C) Unlabeled VP1u was internalized for 30 min at 37°C and detected by Western blotting. (D) Binding assay with ΔC128 and ΔN29/ΔC128 VP1u. Cells were incubated with unlabeled VP1u for 1 h at 4°C and subsequently washed. Bound VP1u was detected by IF with an anti-FLAG antibody.