Coimmunoprecipitation of protein complexes.

Tn5B1 cells or polyclonal stably transformed cells expressing HA-tagged KLC and KHC (50 ×10⁶) were infected with either ac141KO-HA-AC141 (5) or WT virus at a multiplicity of infection (MOI) of 10. Infected cells were harvested at 24 h p.i. (hpi) and resuspended in 1.25 ml of either HEPES lysis buffer (15 mM HEPES, pH 7.6, 10 mM KCl, 0.1 mM EDTA, 0.5 mM EGTA 1 mM dithiothreitol, 1% protease inhibitor cocktail [Invitrogen]) or EBC lysis buffer (50 mM Tris-HCl, pH 8.0, 120 mM NaCl, 0.5% Nonidet P-40, 0.2 mm sodium orthovanadate, 1% sodium fluoride, 1% protease inhibitor cocktail) (5, 45). The lysates were passed through a French press twice at 1,000 lb/in² and centrifuged at 6,000 × g for 20 min at 4°C. The lysates were incubated with either an EZ^view Red anti-HA affinity gel conjugated to an anti-HA monoclonal antibody (Sigma-Aldrich) or polyclonal AC141 antibody (6 μg) diluted in 200 μl 1× phosphate-buffered saline (PBS; 136.66 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄). The mixture was incubated overnight at 4°C on a rotor wheel. The AC141 antibody and lysate mix was further incubated with protein G Dynabeads (Invitrogen) at 4°C on a rotor wheel for 2 h. Red affinity beads or Dynabeads bound to lysate were washed three to five times with either NETN wash buffer (20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5% NP-40, 150 mM NaCl) or Tris-buffered saline (TBS) wash buffer (50 mM HEPES, pH 7.6, 150 mM or 500 mM NaCl, 0.1% Triton X-100). The beads were eluted with 180 μl of elution buffer (100 mM glycine-HCl, pH 2.2), and the pH was raised with 1.5 M Tris-HCl, pH 8.8, to a final pH of 8. The eluent volume was vacuum concentrated to 60 μl and mixed with 20 μl of 4× protein sample buffer (PSB; 277.8 mM Tris-HCl, pH 6.8, 44.4% [vol/vol] glycerol, 0.02% bromophenol blue, 4% β-mercaptoethanol, 1% protease inhibitor cocktail [Sigma-Aldrich]). The sample was boiled for 10 min and subjected to SDS-polyacrylamide gel electrophoresis (PAGE) using Mini-Protean TGX stain-free gels (Bio-Rad), and the proteins were examined by Western blotting.