Phosphorylation of ERK- and DIA-MS–based interaction assay. (A) The activation of ERK phosphorylation of neutrophils from Mmd2^+/+, Mmd2^−/−, Mmd2^A117V/+, Mmd2^A117V/A117V, Mmd2^R127P/+, Mmd2^R127P/R127P, and Mmd2^V100L/V100L mice. Neutrophils were stimulated with fMLP for 0, 3, 5, and 10 min, and the cell lysate was used in immunoblotting, using antibodies against phosphorylated ERK and total ERK. The same experiment was carried out at least three times, and one set of representative data is shown. Data are presented as mean ± SD. **P < 0.01 and ***P < 0.001 with one-way ANOVA with Tukey–Kramer test. ns = not significant n = 3. 0 min versus 10 min in Mmd2^+/+; P < 0.0001. 10 min in Mmd2^+/+ versus 10 min in Mmd2^A117V/+; P = 0.0816. 10 min in Mmd2^+/+ versus 10 min in Mmd2^R127P/+; P = 0.3123. 0 min versus 10 min in Mmd2^+/+; P < 0.0001. 10 min in Mmd2^+/+ versus 10 min in Mmd2^−/−; P < 0.0001. 10 min in Mmd2^+/+ versus 10 min in Mmd2^A117V/A117V; P = 0.0033. 0 min versus 10 min in Mmd2^V100L/V100L; P < 0.0001. 0 min versus 10 min in Mmd2^A117V/A117V; P = 0.0006. 0 min versus 10 min in Mmd2^R127P/R127P; P = 0.0006. (B) Mass spectrometry of interacting Ras proteins after immunoprecipitating MMD2. Binding of RAS to mutated MMD2. (C) To examine the interaction between MMD2 and RAS, co-immunoprecipitation analysis was performed by co-expressing Flag-tagged MMD2-WT or MMD2-mut with Myc-tagged NRAS or HRAS. As a control for co-IP, a sample immunoprecipitated with an anti-HA antibody was included. Additionally, co-immunoprecipitation was performed with RelA, which has not been reported to interact with RAS, as a negative control. MMD2-derived band is highlighted with blue arrowhead. Source data are available for this figure: SourceData F5.