Gpadh

Treated cells were lysed on ice for 30 min and the supernatant cleared by centrifugation at 12000 g for 15 min at 4 °C. Equal amounts of protein per gel lane were separated by 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% skim milk for 1 h in Tris-buffered saline containing 0.1% Tween 20 (TBST) at room temperature (RT), and then incubated overnight at 4 °C in primary antibodies. The blotted membranes were washed 3 times with TBST and probed for 1 h with appropriate HRP-conjugated secondary antibodies (Cell Signalling Technology). The immunoreactive bands were visualized using an enhanced chemiluminescence detection system (Bio-Rad). The relative intensities of bands were measured by ImageJ software. CYP1A1, 12-LOX, phosphorylated-p50, p50, phosphorylated-JNK and total JNK antibodies were purchased from Abcam. Antibodies against phosphorylated-c-jun, phosphorylated-c-fos, phosphorylated-p65, phosphorylated-ERK_1/2, phosphorylated-p38, c-jun, c-fos, p65, ERK_1/2, p38, GPADH and β-actin were purchased from Cell Signalling Technology. All antibodies mentioned above are diluted 1:1000 for western blot analysis.

Whole-cell lysates were prepared using 2% SDS, sonicated and centrifuged (15000 g) at 4°C for 15 min. The supernatants were boiled for 5 min and size-fractionated by SDS/PAGE (10% acrylamide). After transferring proteins on to nitrocellulose filters, the blots were incubated with primary antibodies recognizing APC, AMER3, SLC9A9, GPADH, β-actin, and factor in the Hippo pathway (Cell Signaling Technology, Beverly, MA, United States); following incubation with secondary antibodies, immunocomplexes were developed by using chemiluminescence.