Fig. 6

DHA decreased the levels of vATPase via ubiquitination in AR42J cells. a Cells were pretreated with the indicated concentrations of DHA for 2 h and were then stimulated with cerulein (10⁻⁸ M) for 30 min. Total cell extracts were prepared and subjected to immunoprecipitation (IP) analysis with anti-vATPase E subunit antibody, followed by western blotting (WB) with an anti-ubiquitin (Ub) antibody. Input was used as a control for protein expression determined by western blotting. The upper lane shows total cell extracts subjected to IP with an anti-vATPase E subunit antibody, followed by WB with an anti-ubiquitin antibody. The lower lane shows the amount of input vATPase E subunit determined with western blot analysis (upper panel). The level of the band densities corresponding to ubiquitinated vATPase E subunit to that of its input control was calculated. The band density level of “None” was set as 100%. Values are expressed as the mean ± S.E. of three/each group. *p < 0.05 vs. cells without any stimulation or treatment, ⁺p < 0.05 vs. cells with cerulein stimulation alone (lower panel). b Cells were pretreated with DHA (50 μM) and the ubiquitination inhibitor, PYR-41 (20 or 50 μM), for 2 h and were then stimulated with cerulein (10⁻⁸ M) for 2 h. Protein levels of the vATPase E subunit were determined by western blotting. Actin was used as a loading control. c Levels of the vATPase E subunit in cytosolic and membrane fractions were determined by western blot analysis. Aldolase A and Na⁺/K⁺-ATPase were used as markers of cytosolic and membrane fractions, respectively