PACS-2 levels are regulated by K48-linked polyubiquitination and proteasomal degradation.

<p>(A) HuH-7 cells were treated with TRAIL (20 ng/ml) in the presence or absence of the SMAC mimetic JP1584 (500 nM) for up to 4 hours. Total cell lysates were analyzed by immunoblot with antisera against total and phospho-JNK (T183/Y185), cIAP-1, cIAP-2 and actin (loading control). (B) HuH-7 cells stably over-expressing HA-PACS-2 or wild-type HuH-7 cells were lysed and subjected to HA pull-down using HA agarose beads. The samples were analyzed by immunoblot with antisera against total ubiquitin. (C) HuH-7 cells over-expressing HA-PACS-2 were lysed and subjected to immunoprecipitation using antibodies against Lys48-linked or Lys63-linked poly-ubiquitin, then analyzed by immunoblot for HA. Total cell lysates show equal expression of HA-PACS-2 in the samples. (D) HuH-7 cells (left panel) or HuH-7, KMCH and Mz-ChA-1 cells transiently transfected with S-peptide/SBP-double tagged PACS-2 (right panel) were treated with MG132 (10 µM) for the indicated times. PACS-2 cellular levels were analyzed by immunoblot in total cell lysates.