Fig. 3.

GRK2 is a proviral host factor that becomes activated within minutes of IAV infection. a–c Effect of GRK2 silencing on the replication of A/WSN/33 (H1N1) (a), A/Udorn/72 (H3N2) (b) or A/FPV/Dobson/34 (H7N7) (c) in A549 cells. At 48 h post siRNA transfection, cells were infected with IAV, supernatants were collected at the indicated times and virus titers were determined by plaque assay. Error bars represent standard deviation from three independent experiments, each performed in triplicates. Statistical significance was determined by unpaired t-test. For all panels, ns (non-significant) = P > 0.05; *P ≤ 0.05; **P ≤ 0.01. d A549 cells were transfected with HA-GRK2 and myc-EGFR-encoding plasmids, serum starved for 16 h before being stimulated with recombinant human epidermal growth factor (hrEGF 100 ng/ml). The phosphorylation status of the indicated proteins was examined using a Zn²⁺ Phos-tag gel. Samples on the right (+LPP) were treated with lambda protein phosphatase (LPP) for 30 min at 30 °C before being run on a Zn²⁺ Phos-tag gel. e Same experimental set up as in panel (d), but cells were infected with A/WSN/1933 with an MOI = 100 PFU/cell. f Hep-2 cells were transfected with HA-GRK2- and myc-EGFR-encoding plasmids and 24 h later stimulated with hrEGF or infected with A/WSN/1933 (MOI = 100 PFU/cell) for 15 min. Cells were fixed and stained using antibodies against the HA-tag (green), EGFR (red) and DAPI (blue) to mark the nuclei. Scale bar corresponds to 10 μm, for zoom images scale bar corresponds to 2.5 μm. g Hep-2 cells were transfected with an HA-GRK2-encoding plasmid and 24 h later infected with A/WSN/1933 (MOI = 100 PFU/cell) for 5, 15 or 30 min. Cells were fixed and stained as in (f). Scale bar corresponds to 10 μm