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. 2016 Apr 11;291(23):12282–12293. doi: 10.1074/jbc.M115.698092

FIGURE 6.

FIGURE 6.

Constitutively active PKD induces cell death, mediated by ASK1 and JNK. A and B, F11 cells were co-transfected with the empty pHA vector (vector) or pcDNA3-constitutively active PKD (caPKD) together with the pcDNA3 vector (vector2), pcDNA3-dominant-negative ASK1 (dnASK1), or pcDNA3-dominant-negative JNK (dnJNK). At 72 h, the cells were harvested for trypan blue exclusion assays (A). The cell lysates were subjected to SDS-PAGE and immunoblot analysis with a PKD antibody or a monoclonal antibody against HA (B). C, F11 cells were co-transfected with pcDNA3-FLAG-JNK1a1 and the empty pHA vector (vector), pRK5-T835M-UNC5C, or pHA-V642I-APP. At 48 h after transfection, the cells were harvested for the preparation of cell lysates. FLAG-JNK1a1 in the lysates was immunoprecipitated with a FLAG antibody (M2) and used for in vitro kinase assays with c-Jun-derived peptide as a substrate. Whole mixtures were subjected to SDS-PAGE and immunoblot analysis with an HA antibody, phospho-c-Jun antibody, phospho-JNK antibody, or FLAG antibody (M2). Similar results were obtained in three independent experiments. ***, p < 0.001; n.s., not significant; WB, Western blot.