Fig. 4. Time course of MVP de novo synthesis induced by IFN-. (A) Hep3B cells were treated for the indicated time intervals with IFN- (250 IU/ml) prior to a 30 minute pulse with [³⁵S]methionine. The upper panel illustrates the overall incorporation of [³⁵S]methionine in cell lysates, the middle panel shows Coomassie Blue staining of the same gel to demonstrate equal loading of the lanes. The lower panel depicts the labeled, immunoprecipitated MVP using the antibody LRP56 (MVP) as compared with an unrelated mouse IgG1 antibody (IgG). Quantification of three experiments by ImageQuant software is shown as a bar chart on the right. The amount of newly synthesized MVP was normalized to the total protein content and subsequently to the overall ³⁵S incorporation. Values are given as mean fold induction compared with untreated control cells. (B) MVP, ICAM-1 and ß-actin expression in Hep3B cells treated with IFN- (250 IU/ml) for the indicated time periods were analyzed in parallel by western blot. All experiments shown in this figure were performed at least twice with comparable results.