FIG 7.
Monoclonal antibodies to NSP2 coimmunoprecipitate NSP5. Mock- and rotavirus-infected cell lysates were prepared in RIPA buffer containing protease inhibitors. (A to C) Aliquots of the lysates were either left untreated (A) or incubated with phosphatase inhibitors (B) or calf intestinal phosphatase (C). Immunoprecipitations (IPs) were performed with MAb dNSP2NT or MAb vNSP2CT. Input lysates and IP samples were analyzed by SDS-PAGE, and Western blots were probed for NSP5 using a PAb guinea pig anti-NSP5 serum. Duplicate gels were probed for NSP2 using PAb anti-NSP2, shown in the bottom blots. Anti-NSP5 and anti-NSP2 polyclonal antibodies were detected with a secondary goat anti-guinea pig IgG antibody conjugated to alkaline phosphatase. The molecular masses (MM) 26-28 kDa indicate the glycosylated, hypophosphorylated forms of NSP5 and MW 32-35 kDa indicates the migration range of hyperphosphorylated NSP5.