FIG. 3.

Time course of NS5A synthesis and phosphorylation. (A) Pulse-labelling of NS5A expressed from the construct pcD3-5A. Hep3B cells were transfected with pcD3-5A and labelled with [³⁵S]methionine for the indicated times. (B) Pulse-chase analysis of NS5A phosphorylation. Cells were transfected as described above, labelled for 15 min with [³⁵S]methionine, and chased for the indicated times as described in Materials and Methods. NS5A was immunoprecipitated with NS5A antiserum, and proteins were loaded onto an SDS–7.5% polyacrylamide gel. NS5A p56 and p58 proteins as well as the precursor proteins NS3-5A, NS4A-5A, and NS4B-5A are indicated by arrows on the right. M, molecular weight marker proteins.