Direct Method by Metabolical Labeling

More direct evidence that cellular actin is ADP-ribosylated in intact cells can be obtained, when cells are metabolically labeled with [ P]orthophosphoric acid or with [2- H]adenine. Radioactively labeled phosphate/adenine is incorporated into cellular NAD which is the cosubstrate for C2l-catalyzed ADP-ribosylation. 

Depending on the cell line, the cells are incubated in phosphate-free medium in the presence of 1 mCi [ PJorthophosphoric acid per ml of medium for 2 to 24 h to allow equilibration of the different phosphate pools. 

After treatment with C2II/C2I (see 11.2.1) the cells are rinsed with ice-cold PBS. 

Cell lysis is performed either directly by addition of Laemmli sample buffer, or in a lysis buffer containing 10 mM of unlabeled NAD. 

Alternatively the cells are metabalically labeled with 50(j.Ci [2- H]-adenine per ml of medium for 16 h (Staddon etal., 1991). Labeled actin can be identified by immunoprecipitation with anti-actin antibody (from Boehringer or ICN) or by immunoblot analysis of cell lysates electrophoretically resolved on 2D gels. 

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