FIG 7.

The interaction between ANP32A and polymerase subsides on an active polymerase. (a to c) HEK 293T cells were transfected with PB1_luc1; PA; 0, 0.625, 1.25, 2.5, 5, or 10 ng PolI 76-nt vRNA; and either chANP32A_luc2 and PB2 627E (a), huANP32A_luc2 and PB2 627K (b), or huANP32A_luc2 and PB2 627E (c). Western blotting assay to show expression of tagged plasmids is shown beneath the graphs. Vinculin was used as a loading control. Gaussia luciferase antibody recognizes both luc1 and luc2. Statistical significance was assessed by one-way analysis of variance, and comparisons were made to sample with no added RNA (leftmost bar). ns, nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (d) HEK 293T cells were transfected with expression plasmids encoding PB1, PB2 627E or PB2 627K, PA, and either chANP32A-FLAG or huANP32A-FLAG in the presence or absence of a PolI plasmid expressing a viral-like RNA. GFP-FLAG was used as a control. Thirty hours after transfection, cells were lysed, FLAG-tagged proteins were immunoprecipitated, and coprecipitation of PB2 was detected using Western blotting. IN, input; PD, pulldown. Results representative of three independent experiments.