Fig. 3

SYT11 stabilizes JNK phosphorylation via SYT11-MKK7-JNK complex formation. A, The interaction of SYT11 with phosphorylated JNK1: MKN1 and HEK293T cells were transfected with Myc-JNK1 or Myc-JNK2 alone or together with GFP-SYT11. Cell lysates were used for an immunoprecipitation assay using an anti-Myc or anti-GFP antibody. B, Binding of SYT11 and mutant JNK1: MKN1 and HEK293T cells were transfected with Myc-JNK1 WT, Myc-JNK1 T183A, or Myc-JNK1 Y185F alone or together with GFP-SYT11. Cell lysates were used for an immunoprecipitation assay using anti-Myc antibody. C, SYT11 interaction with MKK7: MKN1 cells were transfected with Myc-MKK7 and GFP-SYT11. Cell lysates were used for an immunoprecipitation assay using anti-Myc. D, JNK1 interaction with MKK7: HEK293T cells were transfected with Myc-JNK1 and HA-MKK7. E, Requirement of MKK7 for interaction between SYT11 and JNK1. F, Requirement of SYT11 for cJun phosphorylation by MKK7. The level of phosphorylation of JNK and cJun was analyzed using the ImageJ software (n = 3). **p ≤ 0.01; *p ≤ 0.05 (Student’s t-test). G, Co-localization of SYT11 with JNK or MKK7 in MKN1 cells. H, Deletion mutants of GFP-SYT11. I and J, HEK293T cells were transfected with the deletion mutants of GFP-SYT11, Myc-JNK1, and HA-MKK7 in the indicated combinations. Cell lysates were immunoprecipitated using anti-GFP antibody. K, Proposed JNK phosphorylation by SYT11 and MKK7