Author: Qiao, Hui; Pelletier, Sandra L.; Hoffman, Lucas; Hacker, Jill; Armstrong, R. Todd; White, Judith M.
Title: Specific Single or Double Proline Substitutions in the “Spring-loaded” Coiled-Coil Region of the Influenza Hemagglutinin Impair or Abolish Membrane Fusion Activity Document date: 1998_6_15
ID: 78fjem8s_18
Snippet: Cells expressing wt-and mutant HA0s were treated with trypsin to cleave HA0. HA-expressing cells were then incubated in MES-saline at the indicated pH value for 15 min at 37 Њ C, reneutralized in MES-saline, pH 7, lysed in cell lysis buffer, and then digested with 0.2 mg/ml proteinase K in lysis buffer with 2 mM CaCl 2 for 30 min at 37 Њ C. The digestion was stopped by adding 0.5 g/ml BSA, 1 mM PMSF, and a protease inhibitor cocktail (Kemble et.....
Document: Cells expressing wt-and mutant HA0s were treated with trypsin to cleave HA0. HA-expressing cells were then incubated in MES-saline at the indicated pH value for 15 min at 37 Њ C, reneutralized in MES-saline, pH 7, lysed in cell lysis buffer, and then digested with 0.2 mg/ml proteinase K in lysis buffer with 2 mM CaCl 2 for 30 min at 37 Њ C. The digestion was stopped by adding 0.5 g/ml BSA, 1 mM PMSF, and a protease inhibitor cocktail (Kemble et al., 1993) . Samples from metabolically labeled cells were then precipitated with the site A monoclonal antibody and analyzed by SDS-PAGE and phosphorimager analysis. Unlabeled samples were analyzed by Western blotting with an anti-HA polyclonal antibody.
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