Fig. 2.

IL-33 induces IRAK-M phosphorylation and binding to PIN1. a GST PIN1 pulldown assay with DC2.4 cell extracts either non-treated or treated with IL-33 (100 ng/ml) or LPS (100ng/ml) for 1 h. The GST-PIN1 bound proteins were eluted using reduced gluthatione and probed for IRAK-M. In the lower panel Coomassie blue staining of the blot shows equal amounts of GST or GST-PIN1 that were used for pull-down. b GST-PIN1 pulldown of DC2.4 cell extracts either treated or not with IL-33, followed by treatment in the absence or presence of calf intestinal alkaline phosphatase (CIP) for 30 min at room temperature before subjecting to GST-PIN1 pulldown. c DC2.4 cells were labeled with 10 μCi/ml {γ-³² P}ATP for 3 h. The cells were washed with fresh medium and treated with 100 ng/ml IL-33 for the indicated times prior to IRAK-M immunoprecipitation. d DC2.4 cells stably expressing IRAK-M were treated with IL-33 and at the indicated time points were subjected to CO-IP using anti-PIN1 antibody and blotted for IRAK-M. e HEK293 cells were transfected with different IRAK-M constructs expressing the N’ terminal domain (aa1-220), the middle portion of the protein (aa220-440) or the C’ terminal domain (aa 440-630), and then treated with IL-33, followed by CO-IP for PIN1. f DC2.4 cells stably expressing IRAK-M were treated with IL-33 and subjected to GST or GST-PIN1 pull-down. The bound proteins were eluted and subjected to IP using IRAK-M antibody. g LC-MS/MS analysis shows phosphorylation of IRAK-M at position Ser110. h WT IRAK-M or its mutants lacking the death domain (IRAK-M ∆DD), lacking the kinase domain (IRAK-M ∆KD), IRAK-M S110A or IRAK-M S467A (where these serine residues were mutated to alanine) were expressed in HEK293 cells, followed by CO-IP for GFP after IL-33 treatment. i IRAK-M, S110E or P111A stably expressing DC2.4 cells were stimulated with IL-33 and PIN1 interaction was monitored by CO-IP experiment as indicated. j HEK293 cells were co-expressed with IRAK-M and GFP, GFP-PIN1, GFP-WW domain or GFP-PPIase domain and, then treated with IL-33 before CO-IP for GFP. k HEK293 cells were co-expressed with IRAK-M and WT PIN1, PIN1 mutant W34A, or PIN1 mutant K63A and then were treated with IL-33 before CO-IP for GFP