Fig. 5

E3 ligase Mind bomb homolog 1 (MIB1) promoted proteasomal degradation of acid ceramidase (ASAH1). A–C Primary pancreatic acinar cells were treated with saline or 200 nM of cerulein plus 10 ng/mL of LPS for 6 h. Cells were lysed, and the lysate was subjected to immunoprecipitation with an anti-ASAH1 antibody. The proteins binding to ASAH1 were denatured and separated by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) gel. A, B The gel was stained with Coomassie brilliant blue (A), and the protein band with square was detected by mass spectrometry (n = 1) (B). The proteins on the gel were transferred to the PVDF membrane and visualized by ECL substrate (C). D, E Primary pancreatic acinar cells were treated with different concentrations of cerulein plus LPS (10 ng/mL) for 6 h (D) or treated with 200 nM of cerulein plus LPS (10 ng/mL) for indicated durations (E); cells were lysed, and cell lysate was examined by Western blotting with indicated antibodies. F, G Lentivirus particles containing nontargeting shRNA or shRNA targeting MIB1 were added to primary pancreatic acinar cells. Cells were lysed, a proportion of cell lysate was analyzed by Western blotting (F), and the other proportion was subjected to immunoprecipitation (G)