Figure 5.

CircWDR37 promotes the binding of PKR to IKKβ and facilitates the interaction of PKR with IκBα thus releasing p65 from inhibitory IκBα. a) Lysates from HEK293T cells co‐transfected with plasmids expressing PKR‐Flag, along with IKKβ‐HA were immunoprecipitated with an anti‐HA antibody (left) or anti‐Flag antibody (right), the precipitates and whole‐cell lysates were then analyzed by western blots with indicated antibodies. b) In vitro purified GST‐PKR protein bound to agarose beads were added to the lysate of HEK293T cells overexpressing IKKβ‐HA. c) The schematic diagram of IKKβ and its recombinant protein truncation proteins. d–f) HEK293T cells transfected with the indicated plasmids were subjected to IP and then analyzed by western blots with indicated antibodies. g) Co‐IP showed the binding of endogenous PKR with p65 proteins in HONE1 and S18 cells. h) In vitro purified GST‐PKR protein bound to agarose beads were added to the lysate of HEK293T cells overexpressing p65‐HA. i) The schematic diagram of p65 and its recombinant protein truncation proteins. j–l) HEK293T cells transfected with the indicated plasmids were subjected to IP and then were analyzed by western blots with indicated antibodies. m) CircWDR37 (0, 1, 3, or 5 µg), PKR‐Flag and IκBα‐myc were co‐transfected into HEK293T cells and processed for IP and were then analyzed by western blots with indicated antibodies. n) CircWDR37 (0, 1, 3, or 5 µg), p65‐Flag, and IκBα‐myc were co‐transfected into HEK293T cells and processed for IP and were then analyzed by western blots with indicated antibodies. o) In vitro purified GST‐PKR^T446DT451D protein first treated with or without alkaline phosphatise, then bound to agarose beads and added to the lysate of HEK293T cells overexpressing IκBα‐myc. p) A schematic model for the function of circWDR37 in PKR‐IKKβ or PKR‐IκBα‐p65 interaction. CircWDR37 promoted PKR interacting to IKKβ. PKR and IκBα competitively interacted with p65, which was also facilitated by circWDR37.