Figure 4.

Newly synthesized HA is a part of large complexes until it assembles into trimers. Influenza-infected CHO15B cells were labeled for 3 min and chased for different time intervals as indicated. The chase was stopped by ice cold PBS containing alkylating agent NEM. The cells in each dish were scraped and divided into two aliquots. One aliquot was cross-linked with DSP as described in Materials and Methods and the other was used as an uncross-linked control. After cross-linking the cells were lysed. The lysates from the crosslinked (lanes 6–10) and the control samples (lanes 1–5) were immunoprecipitated with anti-NHA1 (Fig. 4 A), anti-calnexin (CNX, Fig. 4 B, top panel), and the trimer specific monoclonal antibody (Fig. 4 B, bottom panel) as described in Materials and Methods. The anti-calnexin and anti-trimer immunoprecipitates were analyzed only in their nonreduced form (Fig. 4 B). The anti-NHA1 immunoprecipitates were analyzed both in the nonreduced (Fig. 4 A, top panel) and the reduced forms (Fig. 4 A, bottom panel). The immunoprecipitates were analyzed by 5% SDS-PAGE and fluorography.