Fig. 3

TACI uses a MyD88-binding site to attach the central domain of mTOR. a Left graphs: structure of His-tagged WT TACI or D1 and D2 TACI deletion mutants. Numbers indicate C-terminal and N-terminal residues. ED extracellular domain, TD transmembrane domain, CD cytoplasmic domain. Right gels: IB of mTOR, MyD88 and TACI following IP with anti-His or irrelevant IgG antibodies of protein lysates from transfected 293 cells expressing WT, D1, or D2 TACI. Red numbers indicate band intensity relative to TACI. b NF-κB luciferase (NF-κB-Luc) reporter assay of 293 cells transfected with WT or mutant TACI shown as in a. Ctrl, 293 cells transfected with an empty plasmid. c IB of mTOR, MyD88, and TACI following IP with anti-His or irrelevant IgG antibodies of protein lysates from transfected 293 cells expressing His-tagged WT TACI or mutant TACI proteins with site-targeted substitutions inhibiting MyD88 binding to the cytoplasmic MBS of TACI, including S231R TACI and C233G TACI. d Left graphs: structure of FLAG-tagged WT MyD88 or D1, D2, D3, and D4 MyD88 deletion mutants. Right gels: IB of MYC and FLAG following IP with anti-FLAG or irrelevant IgG antibodies of protein lysates from co-transfected 293 cells expressing FLAG-tagged WT, D1, D2, D3, and D4 MyD88 together with MYC-tagged mTOR. Red arrows indicate nonspecific bands. e NF-κB-Luc reporter assay of 293 cells co-transfected with WT TACI and WT or mutant MyD88 shown in d for 24 h. Ctrl, 293 cells transfected with an empty plasmid. f Left graphs: structure of MYC-tagged WT mTOR or D1, D2, and D3 mTOR deletion mutants. FAT stands for FRAP, ATM, TRRAP domain. Right gels: IB of MYC and HA following IP with anti-HA or irrelevant IgG antibodies of protein lysates from co-transfected 293 cells expressing MYC-tagged WT, D1, D2, and D3 mTOR together with HA-tagged MyD88. Data represent one of three experiments with similar results (a, c, d, f) or summarize three experiments (b, e). Error bars, s.e.m.; *p < 0.05, *p < 0.01 (two-tailed Student’s t test)