Fig. 5. MTK1 mediates delayed and sustained activation of SAPKs by oxidative stress.

(A) Parental HEK293 cells (WT), MTK1 knock-out cells (MTK1^KO), and MTK1^KO cells reexpressing Myc-MTK (MTK1^KO + Myc-MTK1) were treated with H₂O₂ (1 mM) for the indicated times. Phosphorylation levels of p38, JNK, MKK3/6, and MKK4 were analyzed by immunoblotting using appropriate phospho-specific antibodies (second, fourth, sixth, and eighth rows). The expression levels of MTK1, p38, JNK, MKK3/6, and MKK4 in cell lysates are also shown (top, third, fifth, seventh, and bottom). (B and D) ASK1 mediates early activation of SAPKs by oxidative stress. HEK293 cells and ASK1-KO cells (clones #1 and #2) were stimulated with H₂O₂ [1 mM for (B) and 0.1 mM for (D)]. Cell lysates were analyzed by immunoblotting for the expression levels and phosphorylation states of the indicated proteins as in (A). (C) MTK1 and ASK1 are major mediators of SAPK activation by oxidative stress. HEK293 and MTK1/ASK1 double-KO cells (clones #1 and #2) were stimulated with H₂O₂ (1 mM). The expression levels and phosphorylation states of the indicated proteins were analyzed by immunoblotting. (E) MTK1 promotes H₂O₂-induced cell death. The indicated cells were treated with H₂O₂ (0.5 mM) for 6 hours. Cell viability was assessed using the CCK8 assay. Data are means ± SEM (n = 3). *P < 0.05; ***P < 0.01.