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. 2021 Aug 31;12(18):1787–1801. doi: 10.18632/oncotarget.28040

Figure 3. 5Z7O inhibits MAP2K7 activity and downstream activation of JNK.

Figure 3

(A) Immunoblot of the MAP2K7 pathway signaling in a panel of T-ALL and non-leukemic LCL cell lines following incubation with 3 μM 5Z7O for 21 hours. (B) Biochemical kinase assay using purified MAP2K7 protein, dead JNK2 substrate, and two methods of kinase activity based on ATP consumption or ADP production. (C) ADP-GLO in vitro kinase activity of purified TAK1-MBP protein following 30-minute pre-treatment with 5Z7O. (D) Immunoblot of KOPTK1 cells treated with 3 μM 5Z7O to evaluate TAK1 inhibition. Diagram indicating effects of 5Z7O in the MKK signaling pathway in T-ALL cells. (E) KOPTK1 cells were treated with sorbitol to evaluate capacity of 5Z7O to inhibit activated MAP2K7. (F) Jurkat and P12-Ichikawa cells transduced with retrovirus expressing the fusion MAP2K7-JNK2 (MKK7-JNK2) protein were treated with 5Z7O to inhibit activated MAP2K7. For phosphorylated ATF2, immunoblot in Jurkat cells required longer exposure.