Dykddddk peptide

The S2 cells expressing the Torso proteins were sonicated, and the membrane fractions were prepared from the cell homogenate by ultracentrifugation at 103,000 × g, at 4 °C for 90 min, and dispersed in resuspension buffer (1:1 v/v solution of HEPES-buffered saline (HBS) and Schneider’s Drosophila medium). After an incubation with or without 10 nM BmPTTH for 10 min, the fractions were solubilized on ice for 15 min in resuspension buffer, containing 1% Triton X-100, 0.5% sodium deoxycholate, 5 mM EDTA, 1 mM PMSF, and a Phosphatase Inhibitor Cocktail (Nacalai Tesque, Inc., Kyoto, Japan). The cell debris was removed by centrifugation at 20,000 × g, at 4 °C for 5 min, and the supernatant was subjected to immunoprecipitation with a monoclonal anti-FLAG antibody (mouse, #M185-3S, Lot. 002, Medical & Biological Laboratories Co., Ltd.) at 4 °C for 1 h, and with Protein A/G PLUS-Agarose (#sc-2003, Santa Cruz Biotechnology, Inc.) for an additional hour. The immunoprecipitates were washed three times with HBS containing 1% Triton X-100 and 0.5% sodium deoxycholate. The precipitated proteins were eluted with 150 μg/ml DYKDDDDK Peptide (Wako Pure Chemical Industries, Ltd.) and subjected to reducing SDS-PAGE. The autophosphorylation was determined by immunoblotting with an antibody that specifically recognizes phosphorylated tyrosine residues.

Whole-cell lysates of HEK293 cells transfected with Flag-tagged GKRP were immunoprecipitated by an anti-DYKDDDDK tag antibody (Wako). The fusion protein was eluted by DYKDDDDK Peptide (Wako) and re-immunoprecipitated by anti-AcK (Cell Signaling Technology). The immunoprecipitate was resolved on a 4–12% gradient SDS-PAGE gel and the protein band was subjected to mass spectrometry analysis by MS Bioworks (Ann Arbor, MI). For mass spectrometry analysis of the compounds in this article, see Supplementary Figure 5.

Anti-MyD88 Ab, anti-phospho IRAK4 Ab, anti-IRAK4 mAb, anti-phospho p65 Ab, anti-phospho ULK1 mAb, anti-ULK1 mAb, anti-phospho AMPKα mAb, anti-AMPKα mAb, anti-LC3B mAb and horseradish peroxidase (HRP)-conjugated Rabbit IgG were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-DYKDDDDK tag mAb, anti-tag antibody magnetic beads, DYKDDDDK peptide, coomassie G-250, compound C and HRP-conjugated anti-β-actin Ab were obtained from WAKO Pure Chemical Industries (Osaka, Japan). Lipopolysaccharide (LPS, Salmonella enterica serotype typhimurium) was purchased from Sigma Aldrich (St. Louis, MO, USA). Pam3CSK4, FLA-ST standard, FSL-1, ssRNA40/LyoVec and poly (I:C) HMW were obtained from InvivoGen (San Diego, CA, USA). Halt protease & phosphatase inhibitor single-use cocktail was purchased from Thermo Scientific. RIPA lysis buffer was obtained from Merck Millipore (Billerica, MA, USA). 3-methyladenine was purchased from Santa cruz Biotechnology (Dallas, TX, USA).