A Schematics showing the cysteine residues covalently linked in the native HA. B Influenza virus HA was immunoisolated from detergent lysates of influenza virus-infected cells with normal (lanes 1–3) or elevated levels of Malectin (stably transfected HEK293Mal cells, lanes 4–6). The folding intermediates IT1, IT2 and NT are shown. The viral, non-glycosylated nuclear protein (NP) is also recognized by the antibody used. Molecular weight markers (200, 116, 97, 66 kDa) are shown on the right. C Assessment of EndoH-sensitivity of oligosaccharides displayed on HA in HEK293 (lanes 1–6) and in HEK293Mal cells (lanes 7–12). Res, EndoH-resistant HA; Sens, EndoH-sensitive HA. The % of EndoH-resistant HA (Res in the upper panel) has been quantified. This gel is representative of a series of experiments in which HA maturation was monitored at different chase times. D Release of HA from Calnexin in HEK293 (lanes 1–3 (non reducing gel); lanes 7–9 (reducing gel)) and in HEK293Mal cells (lanes 4–6 (non reducing); lanes 10–12 (reducing)). Calnexin and associated substrates have been immunoisolated from the lysate of 200'000 cells. Lane αHA shows the ratio of IT1, IT2, NT in infected cells after 2 min chase and the mobility of NP, which does not co-immunoprecipitate with Cnx (specificity control). Viral proteins in the lane αHA have been immunoisolated from the lysate of 20'000 cells. Molecular weight markers (116, 97, 66 kDa) are shown on the right. E Same as D for endogenous Calnexin substrates. Select polypeptides are shown with A–I. F Same as D for ectopically expressed NHK. G Same as D for ectopically expressed α1AT. H Same as D for Malectin-associated HA. The specificity of the anti-Malectin immunoprecipitation is confirmed by the low cross reactivity in infected cells that did not contain ectopically expressed Malectin (lanes 1–3) and by the lack of co-precipitation of the non-glycosylated NP (lane 1) with Malectin (lanes 4–6). Quantitations of HA release from Calnexin (upper panel, reducing gel, )) and from Malectin (lower panel, gel in ) are shown. Ectopically expressed Malectin-HA and the associated influenza virus HA have been immunoisolated from detergent extracts with an antibody to the HA-tag sequence (YPYDVPDYA). This sequence is not present in the X-31 influenza virus HA (as confirmed by the lack of influenza virus protein in the immunoisolates of cells not expressing Malectin-HA, lanes 1–3). I Same as C for the labelled HA immunoisolated from cells after 20 min chase (lanes 1–2) and for Malectin-associated HA (lanes 3–4) after 20 min chase. K HA immunoisolated after 2 min chase from cells in the absence (Mock, lane 1) or in the presence of 1 mM Castanospermine (Cst, lane 4). The same for Calnexin-associated HA (Mock, lane 2; Cst, lane 5). The same for Malectin-associated HA (Mock, lane 3; Cst, lane 6). The analysis was also performed for cells solubilised after 20 min chase (lanes 7–12). Since Cst inhibits removal of glucose residues from HA-bound oligosaccharides, HA has slower electrophoretic mobility in lanes 4–6 and 10–12. The experiments shown in panels I–K have been performed in HEK293Mal cells.