Figure 5
Effect of the H2A4 3' UTR on regulation of CAT expression. (A) Protein extracts from 35S-labelled L. infantum promastigotes transfected with pXCAT3'H2A and treated with 5 mM HU either for 12 h (lane 0) or at the indicated time period (in h) after removal of the drug, were used to immunoprecipitate CAT. After immunoprecipitation, protein samples were separated by SDS/PAGE and transferred on to a nitrocellulose membrane. Authoradiography (De novo panel) or Western blot with anti-CAT antibodies to reveal the total amount of CAT present in the samples (TP panel) is shown. (B) Densitometric analysis showing the ratio between de novo synthesis of CAT and total amount of CAT (columns). The percentage of cells in the S phase is also included (black line). The assay was repeated twice with similar results. (C) Northern blots prepared with RNA samples separated by sucrose gradients from promastigotes transfected with pXCAT3'H2A and treated 12 with HU or 3 h after drug removal were sequentially hybridized with the CAT and the H4 probe. The autoradiographic exposure of the blots and the densitometric analysis is shown. Data correspond to one representative experiment of three independent assays with similar results.