Englerin A Modulates Stress Signaling Pathways

A498 cells were plated in complete RPMI at 1.2 x 10⁶ and 2.0 x 10⁶ cells per T-75 flask for control and treated cells, respectively. After allowing cells to attach overnight, cells were treated with 0.1% DMSO or 100 nM englerin A for 3, 8, and 24 h. Cell extracts were prepared and Western Blot analysis was conducted as described previously [22 (link)]. Antibodies against MKK4, p-MKK4, eIF2α, p- eIF2α, p-JNK, and B-actin were obtained from Cell Signaling Technology (Danvers, MA) and that against JNK was obtained from Abcam (Cambridge, MA). Except for anti-JNK (diluted 1:2000), all antibodies were diluted 1:1000. Bands were visualized using SignalFire Plus ECL enhanced chemiluminescent substrate (Pierce) and exposed to HyBlot CL film (Denville Scientific). The film was developed with a Kodak film developer.