Figure 5

Formation of soluble Dd-HAWW complexes in insect cells. (A, B) HF cells were infected with the baculovirus co-expressing Dd and HAWW_5 at MOI 5 or (C) by the baculoviruses expressing Dd and HAWW_5 separatel. After 48 h cells were collected and lysed in hypotonic buffer. Clarified lysates were fractionated on 15–40% sucrose density gradients. Gradient fractions were resolved by SDS-PAGE and transferred onto PVDF membrane. The position of HAWW_5 is indicated by dots. (A) Proteins were stained with Ponceau S or (B) revealed with anti-HA antibody. M – molecular weight markers, lanes 1 to 12 – gradient fractions. (C) Insect cells were infected with the recombinant baculoviruses at a ratio of 1 (Dd) to 10 (HAWW_5). Proteins were analyzed as above by Western blot performed with anti-HA antibody. Full length HAWW_5 is marked with a black dot, tentative HA2WW and HA1 are in brackets and the unknown HAWW fragment is marked with question mark.