Fig. 8
FBXL12 mediated ALDH1A1 ubiquitination. (A) Western blotting analysis of ALDH1A1 protein levels in PANC-1, Capan-2 and AsPC-1 cells after treatment with MG132 at 10 µM for the indicated times. (B) Western blotting analysis of ALDH1A1 protein levels in PANC-1, Capan-2 and AsPC-1 cells after treatment with the protein synthesis inhibitor cycloheximide at 10 µM for the indicated times. (C) Statistical analysis of ALDH1A1 protein levels in PANC-1, Capan-2 and AsPC-1 cells after treatment with MG132 at 10 µM for the indicated times. (D) Statistical analysis of ALDH1A1 protein levels in PANC-1, Capan-2 and AsPC-1 cells after treatment with CHX at 10 µM for the indicated times. (E) Western blotting analysis of ALDH1A1 protein levels in FBXL12-knockdown PANC-1, Capan-2 and AsPC-1 cells after treatment with CHX at 10 µM for the indicated times. (F) Statistical analysis of ALDH1A1 protein levels in shRNA-induced FBXL12-knockdown cells after treatment with CHX at 10 µM for the indicated times. (G) Immunofluorescence analysis of ALDH1A1 colocalization with FBXL12 in PANC-1 and Capan-2 cells. (H) Analysis of the interaction between FBXL12 and ALDH1A1 after transfection with the pCMV-Flag-FBXL12 vector by coimmunoprecipitation with an anti-Flag antibody followed by Western blotting analysis. (I) Analysis of the interaction between FBXL12 and ALDH1A1 after transfection with the pCMV-HA-ALDH1A1 vector by coimmunoprecipitation with an HA antibody followed by Western blotting analysis. (J) Analysis of ALDH1A1 ubiquitylation after FBXL12 knockdown or overexpression by coimmunoprecipitation. (K) Analysis of the ubiquitylation of ALDH1A1 by coimmunoprecipitation with an anti-ubiquitin antibody followed by Western blotting analysis with antibodies against the indicated proteins