FIGURE 3.

T231A/S232A double mutation disrupts the interaction of mRIP3 with mMLKL without affecting its kinase activity. A, empty vector, FLAG-mRIP3 wild-type, or T231A, S232A, 2A mutants were co-transfected with HA-mMLKL in 293T cells, respectively. 18 h later, cells were lysed and the lysates were subjected to immunoprecipitation with anti-FLAG beads. The input and immunoprecipitates (IP) were immunoblotted with anti-HA and anti-FLAG antibodies. B, the experiments were performed as in A except that HA-mMLKL was replaced by HA-mRIP1. C, 293T cells were transfected with empty vector, FLAG-mRIP3 wild-type, or FLAG-mRIP3 mutants as indicated. 36 h later, FLAG-mRIP3 wild-type and mutants from cell lysates were immunoprecipitated with anti-FLAG beads. The immunoprecipitates were subjected to in vitro kinase assay with [γ-³²P]ATP using MBP as substrate. The amount of input RIP3 protein was analyzed by immunoblotting with anti-FLAG antibody. mRIP3 kinase-dead mutant D143N was included as a negative control.