Figure 7

Characterization and NiV infection of EB2- and ΔcEB2-expressing PAEC. (A) EB2 and ΔcEB2 were immunoprecipitated from PAEC-EB2 and -ΔcEB2 cell lysates, separated on a SDS gel and analyzed by Western blot analysis using an EB2-specific antibody. (B) Stably EB2- and ΔcEB2-expressing PAEC were seeded on permeable filter supports. After 7 days, apical and basolateral surfaces were stained with EphB4/Fc and rhodamine-conjugated secondary antibodies. After fixation and permeabilization, cells were incubated with anti-VE-cadherin antibodies (VE-Cad) and FITC-conjugated secondary antibodies. (C) PAEC-EB2 and -ΔcEB2 were incubated with recombinant EphB4/Fc for 1 h at 37°C to allow binding and endocytosis to occur. Surface-remained EphB4/Fc was detected with rhodamine-conjugated secondary antibodies (surface) and internalized EphB4/Fc was stained after fixation and permeabilization by FITC-conjugated secondary antibodies (intracellular). (D) EB2- or ΔcEB2-expressing PAEC were infected with NiV at a MOI of 1. At 24 h p.i., immunostaining was performed as described in the legend to Fig. 4A.