Fig 2. Gaussia princeps luciferase-based FluPol—CTD binding assay.

A-B. G2-CTD was expressed by transient transfection in HEK-293T cells together with PB2, PB1 and PA of FluPol_B (B/Memphis/13/2003, blue bars) or FluPol_A (A/WSN/1933, grey bars). Either PB2 (A, hatched bars) or PA (B, filled bars) were C-terminally tagged in frame with G1. As controls, the wild-type (wt) PB1 was replaced by the catalytic inactive PB1 D444A D445A mutant (i) or was omitted (-). Luciferase activities were measured in cell lysates at 24 hpt. Normalised luciferase ratios (NLRs) were calculated as described in the Materials and Methods section. The data shown are the mean ± SD of at least three independent experiments performed in technical triplicates. **p ≤ 0.002; ***p ≤ 0.001 (two-way ANOVA; Dunnett’s multiple comparisons test). C-D. The CTD binding of FluPol_A mutants PA K289A and R638A was investigated. HEK-293T cells were transfected as described in (A) and (B), respectively. Relative Light Units (RLUs) are expressed as percentages relative to the FluPol_A PA wt. The data shown are the mean ± SD of three independent experiments performed in technical triplicates. **p ≤ 0.002; ***p ≤ 0.001 (one-way ANOVA; Dunnett’s multiple comparisons test). The dotted line labelled “Ctrl” indicates the background signal i.e. the sum of the luminescence activities measured in control samples co-transfected with either the FluPol-G1 and G2 plasmids, or the G1 and G2-CTD plasmids. E. Schematic representation of the CTD constructs used in (F) and (G): the wild-type G2-CTD (wt, top) and the G2-CTD in which all serine 5 residues were replaced with an alanine (S5A, bottom). F-G. The interaction of the wt or the S5A mutated CTD to FluPol_B (F) or FluPol_A (G) was investigated by transient transfection in HEK-293T cells as described in (A-B). The data shown are the mean ± SD of four independent experiments performed in technical triplicates. *p ≤ 0.033; ***p ≤ 0.001 (two-way ANOVA; Sidak’s multiple comparisons test). In parallel, cell lysates were analysed by western blot using antibodies specific for the pS5 or pS2 CTD, G.princeps luciferase (Gluc) and tubulin. The slow- and fast-migrating bands detected with the pS2 CTD antibody, which likely correspond to the hyperphosphorylated and hypophosphorylated forms of the CTD, respectively, are indicated by a star and a triangle, respectively. The smeared signal in the G2-S5A-CTD samples likely corresponds to the detection of phosphorylation intermediates.