(a) Western blot analyses of total, cytoplasmic, or nuclear A549 cell extracts 0, 6, and 12 hr post infection (HPI). hnRNP K was analysed via traditional SDS-PAGE or Phos-tag to separate phosphorylated isoforms of hnRNP K. (b) Approximately 100% of A549 cells were infected with influenza virus (WSN) for 5 hr and then subjected to immunofluorescence microscopy to detect SC35, hnRNP K, and viral proteins. Nuclear speckles were marked with anti-SC35 antibody. Scale bar = 10 µm. (c) Voxel outside surface generated for nuclear speckles was set to 0 for hnRNP K channel to visualize hnRNP K at nuclear speckles. The marked region is enlarged. (d) Quantification of hnRNP K at nuclear speckles (NS) was performed using the Imaris software. hnRNP K intensity sum at nuclear speckles was normalized to the intensity sum in the nucleus. Values are means ± s.d. measured in 35 mock and 35 WSN infected cells from biological triplicates. ** unpaired, two-tailed t-test p<0.01. (e) Quantification of nuclear speckle volume was performed using the Imaris software. Surface was generated using fluoresce signal from immunostained SC35 proteins to measure nuclear speckle volume. Values are means ± s.d. measured in ≥35 mock and WSN infected cells from biological triplicates. * unpaired, two-tailed t-test p≤0.05.