FIGURE 4.
Bim binds PACS-2 and is required for recruitment of PACS-2 and activated Bax to lysosomes. A, His6-PACS-2FBR38–217 was mixed with GST-Bax, GST-BimEL, or GST alone. GST proteins were captured, and bound His6-PACS-2FBR38–217 was detected by immunoblot analysis. B, Huh-7 cells transfected with scramble siRNA or with a Bim siRNA were treated with TRAIL (10 ng/ml) for the indicated times. Lysosomal fractions were obtained and analyzed by immunoblot analysis. LAMP 2, lysosomal marker. C, KMCH cells and KMCH cells transfected with a Bim siRNA were treated with TRAIL (10 ng/ml) for the indicated times. Lysosomal fractions were obtained and analyzed by immunoblot analysis. D, Huh-7 cells transfected with scramble siRNA or with a Bim siRNA (siBIM) were treated with TRAIL (10 ng/ml) for 3 h, fixed, and analyzed by immunofluorescence for activated Bax (green) and nuclei (DAPI, blue). Bottom panel, quantification of the percent of cells containing activated Bax expressed as percent of total number of cells per field. Inset, efficiency of Bim knockdown was verified by immunoblot analysis. E, Huh-7 HA-PACS-2 transfected with scramble siRNA or with a Bim siRNA were treated with TRAIL for the indicated times. Cell lysates were collected, HA-PACS-2 was immunoprecipitated (IP) from the cell lysates, and associated proteins were identified by immunoblot analysis. F, scramble-transfected HA-PACS-2 Huh-7 cells or HA-PACS-2 cells transfected with a Bax shRNA were treated with TRAIL (10 ng/ml) for 2 h. HA-PACS-2 was immunoprecipitated from the cell lysates, and interacting proteins were detected by immunoblot analysis. Top panel, Bax expression in scramble and Bax shRNA knockdown cells.