Fig. 2

MIEN1 has a functional immunoreceptor tyrosine based activation motif. a Schematic representation of the ITAM and CAAX-prenylation motif on MIEN1 protein and different site directed mutants used in the current study. Tyrosine residues in ITAM domain were mutated to phenylalanine (F) and cysteine residue of the CAAX-prenylation site was mutated to serine (S). b NIH3T3 cells were transfected with GFP fused MIEN1^WT, MIEN1^Y39F, MIEN1^Y50F and MIEN1^Y39/50F constructs followed by immunoprecipitation using GFP antibody. The tyrosine phosphorylation of MIEN1 was detected using a generic phospho-tyrosine antibody. c NIH3T3 cells were transfected with empty GFP, MIEN1^WT and MIEN1^C112S constructs. MIEN1^WT transfected cells were either treated with DMSO (vehicle control) or geranylgeranyl transferase I (GGTI) inhibitor and then immunoprecipitated using GFP antibody followed by immunoblotting with phospho-tyrosine antibody. The total MIEN1 protein used for immunoprecipitation was indicated as input