The Anaphase-Promoting Complex Protein 5 (AnapC5) Associates with A20 and Inhibits IL-17-Mediated Signal Transduction (original) (raw)
Figure 4
The IL-17R-A20 signaling complex involves AnapC5 rather than Itch and TAXBP1. A. AnapC5 but not AnapC7 associates with A20.
HEK293T cells were co-transfected with AnapC5 [short (s) and long (l) forms] or AnapC7 (both Myc-tagged) together with A20. Lysates were subjected to IP with anti-Myc Abs and blotted for A20 or Myc. WCL were blotted for A20. Migration of protein size markers is indicated. B. AnapC5 associates with endogenous A20. HEK293T cells were transfected with Myc-tagged AnapC5. Lysates were subjected to IP with anti-Myc Abs and blotted for A20 or Myc. WCL were blotted for β-tubulin as a loading control. C. Knockdown of Itch and TAXBP1 does not impact IL-17R mediated signaling. ST2 cells were transfected with the indicated siRNAs, stimulated with IL-17 for 24 h, and mRNA expression was assessed by qPCR. n.s., not significant. *p<0.05 by ANOVA and post-hoc Tukey’s test compared to unstimulated controls. ‡p<0.05 by Chi Square comparing experimental replicates. D. Efficient knockdown of Itch and TAXBP1. mRNA from the samples in panel B were assessed for Itch and TAXBP1 expression by qPCR. *p<0.05 by ANOVA and post-hoc Tukey’s test compared to mock-transfected controls. ‡p<0.05 by Chi Square comparing experimental replicates. All data are representative of at least two independent experiments.