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. Author manuscript; available in PMC: 2010 Jul 1.
Published in final edited form as: Biochim Biophys Acta. 2009 Apr 22;1793(7):1218–1227. doi: 10.1016/j.bbamcr.2009.04.006

FIGURE 3. CerS1 degradation is ubiquitination-mediated.

FIGURE 3

HEK 293 cells stably transfected with FLAG-CerS1 (denoted FLAG-CerS1) were treated with 50 µM cisplatin or 2.5 µM doxorubicin for 6 h, with or without pretreatment with 20 µM MG132 for 1 h. Vector control cells (V) are HEK 293 cells stably transfected with empty vector. A) Cells were extracted with non-denaturing buffer containing Digitonin (Materials and Methods), and equal amounts of extracted protein were immunoprecipitated with anti-FLAG mAb and captured with protein A agarose beads. The immunoprecipitates were then subjected to immunoblot analysis, using mouse anti-ubiquitin mAb P4D1 (upper panel), or anti-FLAG mAb to show that the 39 kDa FLAG-CerS1 protein was indeed immunoprecipitated (lower panel). B and C) The cells were extracted under strongly denaturing conditions to avoid co-Immunoprecipitation proteins. Following Immunoprecipitation with anti-FLAG mAb and protein A agarose, the beads were washed in chaotropic 0.5 M LiCl to dissociate any further associated proteins (see Materials and Methods). The immunoprecipitates were then subjected to immunoblot blot analysis, using mouse anti-ubiquitin mAb P4D1 (B), or anti-FLAG mAb (C). The experiments were repeated 2 times with similar results. (Ub)n-CerS1 – ubiquitinated CerS1, IP - immunoprecipitation, WB - Western blot.