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. 2020 Sep 22;48(18):10479–10499. doi: 10.1093/nar/gkaa765

Figure 4.

Figure 4.

hnRNP A1 post-translational modifications impact on HIV-1 IRES activity. (A) hnRNP A1-HA mutants for the phosphorylation sites S4/S6 and S199, or methylation sites R218/R225 were generated by site-directed mutagenesis of the wt-hnRNP A1-HA template plasmid. (B–E) HEK 293T cells were co-transfected with the dl HIV-1 IRES (300 ng) together with each hnRNP A1-HA (500 ng) plasmid. Total protein extracts were prepared 24 h post-transfection. (B, D) Western blots were performed to detect the expression level of total hnRNP A1 and hnRNP A1-HA proteins using GAPDH as a loading control. (C, E) RLuc and FLuc activities were measured, and results are presented as RTA, relative to the activities in the presence of the wt-hnRNP A1-HA, set to 100%. Values shown are the mean (±SEM) for three independent experiments, each performed in duplicate. Statistical analysis was performed by an unpaired two-tailed t-test (*P<0.05). The relative level of total hnRNP A1 protein, PRMT5 and SDMA (%) in (B, D) was estimated based on the intensity of immunoreactive bands by image-J.