Figure 5.

(G2019S) LRRK2 enhances the phosphorylation of MKK4 at Ser²⁵⁷. (a) Western blot study using anti-HA antiserum showed that a similar protein level of HA-tagged wild-type or mutant (G2019S) LRRK2 was stably expressed in HEK 293 cells. (b) FLAG-tagged MKK4 was transiently transfected into control 293 cells or HEK293 cells stably expressing HA-tagged wild-type or mutant (G2019S) LRRK2. Then, FLAG-tagged MKK4 was immunoprecipitated by anti-FLAG antibody, and phosphorylated MKK4 at Ser²⁵⁷ serine was visualized by performing immunoblotting analysis using anti-phospho-MKK4^Ser257 antibody. As a negative control, addition of FLAG peptide completely blocked immunoprecipitation of FLAG-tagged MKK4. Compared with control 293 cells or HEK 293 cells expressing wild-type LRRK2, immunoprecipitated protein level of phospho-MKK4^Ser257was significantly increased in 293 cells expressing mutant (G2019S) LRRK2. Note that anti-FLAG antibody also co-immunoprecipitated HA-tagged wild-type or (G2019S) LRRK2. Each bar shows the mean±S.E. value of five experiments. *P<0.01 compared with control 293 cells