Renilla luciferase kit

293 T cells were transfected with PB1^luc1 (25 ng), either PB2 627E or PB2 627K (25 ng), PA (12.5 ng) and chANP32A^luc2, chANP32AN129I^luc2, chANP32B^luc2 or chANP32B₃₃^luc2 (12.5 ng). For split luciferase assays measuring histone interaction, 50 ng of either chANP32A^luc2, chANP32AN129I^luc2, H4^luc1 or H3^luc2 were transfected into 293 T cells. Control samples assessed the interaction between H4 or PB1^luc1 and an untagged luc2 construct or the appropriate ANP32A^luc2 construct and an untagged luc1 construct. All other components transfected into control samples remained consistent with those transfected in with the interacting proteins of interest. 24 hr after transfection, cells were lysed in 50 ul Renilla lysis buffer (Promega) for one hour at room temperature. Gaussia luciferase activity was then measured from 10 ul of lysate using the Renilla luciferase kit (Promega) with a FLUOstar Omega plate reader (BMG Labtech). Normalised luminescence ratios were calculated by dividing the luminescence measured from the interacting partners by the sum of the interaction measured from the two controls for each sample (Figure 6—figure supplement 1) as previously described (Cassonnet et al., 2011 (link)).

For measuring ANP32-influenza-polymerase interactions, eHAP DKO cells were seeded into 48-well plates and transfected with pCAGGS expression plasmids encoding Tky05 PB1-Gluc1 (0.04 µg), PA (0.04 µg), PB2 (0.04 µg), and the indicated ANP32-Gluc2 construct (0.04 µg). Control conditions contained pCAGGS-Gluc1 and untagged PB1, or pCAGGS-Gluc2 and untagged ANP32, with all other components remaining constant. Twenty-four hours after transfection, cells were lysed in 60 µl Renilla lysis buffer (Promega) for 1 h at room temperature with vigorous shaking. Gaussia luciferase activity was assayed using the Renilla luciferase kit (Promega). Injection of substrate and measurement of bioluminescence were carried out using the FLUOstar Omega plate reader (BMG Labtech). Normalized luminescence ratios (NLR) were calculated by dividing the signal from the potential interacting partners by the sum of the two controls^{70 (link)}.
For measuring polymerase dimerization, eHAP TKO cells were transfected with pCAGGS expression plasmids encoding Tky05 PB1-Gluc1 (0.04 µg) or PB1-Gluc2 (0.04 µg) both codon optimised for gene expression in H. sapien cells, as well as PA (0.08 µg), PB2 (0.08 µg). The assay was then conducted as detailed above.

pCAGGS expression plasmids encoding A/Tky/5092/91 (H5N1) PB1_luc1, PB2 627E or 627K, PA, and ch- or huANP32A_luc2 were transfected into 293T or eHAP1 cells at a ratio of 2:2:1:1. Control samples included luc1 and untagged PB1 or luc2 and untagged ANP32A, with all other components remaining constant between all samples (Fig. 1b). Where other polymerase components were luc tagged, equivalent appropriate controls were used.
Twenty-four hours after transfection, cells were lysed in 50 μl (48-well plate) or 100 μl (24-well plate) Renilla lysis buffer (Promega) for 1 h at room temperature. Gaussia luciferase activity was assayed using the Renilla luciferase kit (Promega). Injection of substrate and measurement of bioluminescence were carried out using the FLUOstar Omega plate reader (BMG Labtech). Normalized luminescence ratios were calculated by dividing the signal from the chosen interacting partners by the sum of the two controls as described in the work of Cassonnet et al. (30 (link)).