Proteomics Analysis of the Nucleolus in Adenovirus-infected Cells

Lam, Yun W.; Evans, Vanessa C.; Heesom, Kate J.; Lamond, Angus I.; Matthews, David A.

For SILAC analysis, prior to infection cells were grown for five rounds of cell division in Dulbecco's modified Eagle's medium containing L-[ 13 C 6 , 15 N 4 ]arginine and L-[ 13 C 6 , 15 N 2 ]lysine (Cambridge Isotope Laboratories) supplemented with 10% dialyzed fetal calf serum (Biowest) to ensure all the cellular proteins were labeled to saturation. Prior to nucleolar isolation, equal numbers of uninfected, unlabeled cells and Ad5-infected, labeled cells were mixed. Nucleoli were then isolated from the mixed cell populations using a standard protocol (35) . Nucleoli were lysed by heating in lithium dodecyl sulfate sample buffer (Invitrogen), and nucleolar proteins were separated by SDS-PAGE on a 4 -12% precast gradient gel (Invitrogen). The gel was fixed, stained with colloidal Coomassie Blue (Invitrogen), and cut into six slices.