Fig 4.
PKC-α/β interact with RIG-I. (A and B) Interaction between PKC-α/β and RIG-I or RIG-I 2CARD. At 48 h posttransfection with Myc-tagged PKC-α, PKC-βII, or PKG together with vector or Flag-RIG-I (A) or with GST or GST-RIG-I 2CARD (B), WCLs were used for IP with anti-Flag (A) or GST-PD (B), followed by IB with anti-Myc (A and B), anti-Flag (A), or anti-GST antibody (B). (C) Interaction between endogenous RIG-I and PKC-α/β. WCLs of HEK293T, A549, or HeLa cells were subjected to IP with an anti-RIG-I antibody, followed by IB with anti-PKC-α, anti-PKC-βI, anti-PKC-βII, anti-PKC-ε, anti-PKC-ζ, or anti-RIG-I antibody. The input (2%) for 293T is shown. (D) RIG-I-PKC-α/β interaction in primary NHLFs. NHLFs were mock infected or infected with SeV (50 HA units/ml) for the indicated hours. WCLs were subjected to IP with an anti-RIG-I antibody, followed by IB with anti-PKC-α, anti-PKC-βII, or anti-RIG-I antibody. (E) PKC-α and PKC-β that interact with RIG-I are phosphorylated at S657 or T500, respectively. HEK293T cells were mock treated or infected with SeV (50 HA units/ml) or ΔNS1 PR8 virus (MOI, 2) for 14 h. WCLs were used for IP with an anti-RIG-I antibody, followed by IB with anti-PKC-α, anti-pS657-PKC-α, anti-PKC-βII, or anti-pT500-PKC-β antibody.