2.7. Western blot

For western blot assay, BXPC‐3 cells were plated in 6 cm dishes(1 × 10⁶ cells/dish) and treated with different concentrations of umbelliprenin or 3‐MA as indicated. After incubation for 24 h, cells were harvested and extracted by RIPA buffer lysis buffer on ice (Beyotime Institute of Biotechnology). Cell lysate was centrifuged at 14,000 rpm for 10 min and the supernatants were collected. The protein concentrations were quantified by the Quick Start Bradford Dye Reagent (Bio‐Rad). Total protein was mixed by adding 6× loading buffer and boiled for 5 min. A total of 30 μg proteins were measured by 4%–12% SDS‐PAGE (Amersham Bioscience). Following incubated with the primary and secondary antibodies, the membranes were detected by ECL (Thermo Scientific) and analyzed on a ChemiDoc XRS+(Bio‐Rad Laboratories, Inc.).