Quantification of IRF3-SUMOylation

To determine SUMOylation of IRF3 in Influenza A infected cells, Global Protein SUMOylation assay kit (Abcam) was used as described by the manufacturer. Briefly, A549 cells were mock-infected or infected with Influenza A/New Caledonia/20/99 (H1N1) or pdm A/H1N1 2009 viruses. Cells were harvested at 12, 16 and 24 hpi, cytoplasmic and nuclear extracts were isolated, as described above. Both subcellular fractions were incubated with the anti-IRF3 rabbit pAb (FL-425) (Santa Cruz Biotechnology, CA, USA) in 8-well assay microwell strips, and SUMO was measured with the anti-SUMO antibody included in the kit by colorimetry (OD measure at 450nm). Positive and negative controls included in the kit were used. To quantify SUMOylation of IRF3, the values from the negative control were subtracted from the values obtained for extracts from mock- infected or viral infected cells and were compared to the level of mock-infected cells. Then calculate percentage of SUMO conjugated using the following formula: Percentage of IRF3-SUMOylation intensity=                   [OD (infected sample - negative control)]                   ___________________________________   x 100                 [OD (Mock-infected sample – negative control)]  The error bars indicate the standard deviation of the duplicates of two independent measurements.