Figure 2. IRAK4-L results in Myddosome assembly and maximal activation of innate immune signaling.

(A) Pathway analysis of enriched genes in AML patients preferentially expressing IRAK4-L (top) or IRAK4-S (bottom). The pathway scores were calculated by NetWalker⁵¹ based on a random walk method⁵². The scores directly reflect the enrichment of the indicated pathways for high (positive scores) or low (negative scores) expression of the IRAK4 Long isoform. The pathways shown here were the highest-scoring pathways. (B) HEK293 cells transfected with vector (pcDNA3.1), or FLAG-tagged IRAK4-L or IRAK4-S for 24 hours and immunoblotted for the indicated NF-κB and MAPK proteins (three independent experiments). (C) Densiometric analysis of panel (B). One-sided t-tests were used for statistical analyses. (D-E) NF-κB (D) and AP1 (E) activation was measured by κB-site and AP1-site containing reporter assays in HEK293 cells transfected with empty vector, IRAK4-L, or IRAK4-S. Values are normalized to Renilla-luciferase and empty vector (1.0) (3 or 6 independent experiments, respectively). Two- sided t-tests were used for statistical analyses. (F) Representative PCR and immunoblot of THP1 cells following knockdown of IRAK4-L and IRAK4-S (shIRAK4-L/S) or just IRAK4-L (shIRAK4-L) (three independent experiments). (G) NF-κB activation was measured in THP1 cells stably expressing a κB-site containing reporter and shRNAs targeting IRAK4-L (left) or IRAK4-L/S (right) after stimulation with PAM3CSK4 (+, 500 ng/ml or ++, 1000 ng/ml). (3 or 5 independent experiments, respectively). Two-sided t-tests were used for statistical analyses. (H) Overview of IRAK4-L and IRAK4-S downstream signaling based on Figure 2A–E. (I) Sequence-based prediction of protein-protein and domain-domain interactions using all isoforms of IRAK1, IRAK4, and MyD88. Superscripts indicate distinct RNA isoforms encoding the indicated long or short proteins. (J) HEK293 cells transfected with HA-MyD88 and either FLAG-IRAK4-L or FLAG-IRAK4- S were immunoblotted for HA-MyD88 after immunoprecipitation of FLAG. (representative of two independent experiments). (K) TF1 and HL60 cells were immunoblotted for IRAK4 with C-terminal antibody after immunoprecipitation of MyD88 (representative of two independent experiments). *, indicate IgG bands. All data represent the mean ± s.e.m.