Fig. 4.

Interaction of RTP4 with STING, MAVS, TBK1, and IRF3 in vitro. The 293T cells (1 × 10⁶) were cotransfected with plasmids encoding RTP4 and STING, MAVS, TBK1, IRF3, or TRAFs (1 μg each) tagged with either HA, MYC, or V5. The tagged molecules were pulled down and detected using anti-tag antibodies. IP, indicating antibody used in immunoprecipitation; lysate, total proteins from cell lysis; β-actin was used as protein loading control. (A–F) The co-IP and protein expression levels as indicated for STING (A), MAVS (B), IRF3 (C), TBK1 (D), TRAF2, TRAF3, and TRAF6 (E), and MDA5/RIG-I (F). (G and H) 239T cells (1 × 10⁷) were transfected with 5 μg of RTP4-HA plasmid only, and anti-TBK1 or anti-IRF3 mouse antibody was used to pull down TBK1 and IRF3, respectively. RTP4 was detected using anti-HA antibody. Mouse IgG was used as control for potential nonspecific binding in the pulldown experiments. Note: The band in the IgG lane in H is likely a IgG heavy chain. (I and J) HA- or MYC-tagged RTP4, TBK1, or IRF3 were synthesized in vitro using a cell-free protein expression system (PURExpress, NEB). Anti-HA or anti-MYC antibodies were then used to pull down RTP4. TBK1 and IRF3 were detected using anti-MYC (I, TBK1) and anti-HA (J, IRF3).