Fig. 3: EBOV VP35 activates PKA via AKIP1 association.

a Lysates of HEK293 cells cotransfected with the indicated plasmids were incubated with/without recombinant His-VP35 for 2 h and then subjected to immunoprecipitation and immunoblotting analysis. b Wild-type (WT) and AKIP1 knockout (KO) HepG2 cells transfected with GFP-VP35 or GFP were treated with FSK (25 μM) for 45 min and were subjected to anti-PRKACA immunostaining (red). Arrow: cells expressing GFP-VP35. c Lysates of HepG2 cells transfected with Flag-vector, Flag-VP35, or Flag-VP35 mutant plasmids were analyzed by immunoblotting using a PKA substrate phosphorylation antibody. d Lysates of WT and AKIP1^−/− HepG2 cells infected with Ad-VP35 or Ad-null (MOI = 10) were subjected to PKA activity assays. Differences between the two groups were evaluated using a two-sided unpaired Student’s t-test. Data were presented as mean ± s.e.m. ns not significant; **P  <  0.01. e Concentrations of cAMP were detected in the lysates of WT and AKIP1^−/− HepG2 cells infected with live EBOV (MOI = 10). Differences between the two groups were evaluated using a two-sided unpaired Student’s t-test. Data from three independent experiments were analyzed, are presented as the means ± s.e.m. (**P < 0.01; ***P < 0.001).