Figure 7.

In the presence of DTT p4a is cleaved to its mature (4a) form. Infected HeLa cells were treated (DTT +) or not treated (DTT +) from 4 h after infection onwards with 5 mM DTT. In each case, when medium with DTT was applied to the cells, freshly prepared DTT was added to the medium just before use. At 5 h after infection the medium was replaced with medium with or without DTT. At 5.5 h after infection the cells were starved in methionine-free DMEM with or without DTT. Pulse-labeling for 15 min (chase 0 h) was at 6 h after infection and consisted of replacing the methionine-free medium by the same medium containing 50 μCi of [³⁵S]methionine with or without DTT. After the pulse, cells were chased for 2 and 3 h (chase 2 and 3 h) in medium with or without DTT that was replaced each hour. At the end of the pulse or chase cell lysates were prepared and 4a and its precursor form (p4a; both indicated) were immunoprecipitated from them. Immunoprecipitates were run on 10% SDS-PAGE and the proteins detected by autoradiography. M-¹⁴C-labeled marker protein of 93 kD.