title: Ube3a-/- TNF treated MEFs
design: Mice  used were all on a C57BL/6 background. The MEFs from null (Ube3a-/-) and  wild-type (Ube3a+/+) 13.5 days old embryos were generated by breeding  Ube3a-/+ mice. For the MEF isolation, embryos from 13.5-day-pregnant mice  were washed with phosphate-buffered saline (PBS). The head and visceral  tissues were removed and the remaining bodies were washed in fresh PBS,  minced using a pair of scissors. MEFs cells were isolated using the Primary  Mouse Embryonic Fibroblast Isolation Kit (Thermo #88279) according to the  manufacturer instructions. Cells were collected by centrifugation (200  g  for 5 min at 4C) and re-suspended in fresh DMEM medium with 15% FBS. 1  106  cells were cultured on 100 mm dishes at 37C with 5% CO2. In this study, we  used MEFs within three to five passages to avoid replicative senescence.  Housing, handling, and experimental procedures were performed in accordance  with the National Institutes of Health guidelines and were approved by the  University of Haifa animal ethics committee.