Fig 1: TRIM11 facilitates the degradation of Daple by inducing ubiquitination.a CNE2 cells stably expressing SFB-TRIM11; whole-cell lysates were obtained by tandem affinity purification of the TRIM11-associated protein complex as described in the “Materials and methods” section, and affinity-purified TRIM11 complexes were visualized by Coomassie brilliant blue staining. TRIM11 and Daple protein bands are indicated by arrows. b, c CNE2-DDP cells were harvested for immunoprecipitation using anti-IgG, anti-TRIM11 or anti-Daple antibodies and then analyzed by western blotting. β-actin was used as a loading control. d CNE2-DDP cells were fixed after cotransfection with Flag-TRIM11 and HA-Daple for 24 h and analyzed by immunofluorescence analysis. Scale bar, 20 µm. e, f Determination of WT TRIM11-mediated (e) or mutant TRIM11-mediated (f) ubiquitinated Daple. Lysates from cells transfected with the indicated constructs plus HA-tagged ubiquitin were immunoprecipitated with S beads, and the immunoprecipitates were analyzed by western blotting using an anti-HA antibody to detect the Daple/ubiquitin complexes. g Determination of the levels of ubiquitinated Daple between WT and mutant K48R ubiquitin. Lysates from cells transfected with the indicated constructs plus HA-tagged ubiquitin were immunoprecipitated with S beads, and the immunoprecipitates were analyzed by western blotting using an anti-HA antibody to detect the Daple/ubiquitin complexes. h Immunoblot analysis of protein extracts of CNE2 or 293T cells transfected with plasmid for HA-Daple and increasing doses of plasmid for Flag-TRIM11. β-actin was used as a loading control. i, j CNE2 cells stably expressing empty vector or TRIM11 were incubated with 20 μg/ml CHX for the indicated times. Cell lysates were then analyzed by western blotting (i), and the densities of the HA-Daple protein bands at each time point were normalized to β-actin and were calculated as percentages (j). Data are the mean ± SD of triplicate samples. **P < 0.01. k The endogenous protein level of Daple was detected after knocking out TRIM11 in CNE1-DDP and CNE2-DDP cells. β-actin was used as a loading control. l Immunoblot analysis of extracts of CNE2 cells transfected with plasmids for HA-Daple or its mutants, together with Flag-TRIM11. β-actin was used as a loading control. m Immunoblot analysis of extracts of CNE2 cells transfected with plasmids for SFB-TRIM11 or its mutants, together with HA-Daple. β-actin was used as a loading control.