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. Author manuscript; available in PMC: 2019 Mar 30.
Published in final edited form as: J Mol Biol. 2018 Feb 6;430(7):1024–1050. doi: 10.1016/j.jmb.2018.01.021

Figure 6. UBE3A promotes the degradation of ASPP2 by the proteasome.

Figure 6

A.UBE3A coimmunoprecipitates ASPP2. HEK 293T cells were transfected with the indicated vectors. 48 hours after transfection the HA-tagged proteins were immunoprecipitated with anti-HA agarose beads. Protein extracts and immunoprecipitates were analyzed by SDS-PAGE and Western blot using antibodies against HA-tag and Actin. HBH-ASPP2 carrying a biotinylation signal in the HBH tag was detected using streptavidin-HRP. HA-UBE3A: UBE3A isoform 1 C820A, catalytically inactive. B. Coexpression of UBE3A reduces ASPP2 protein levels. HEK 293T cells were transfected with the corresponding vectors. 48 hours post transfection the cells were harvested and the protein extracts were analyzed by SDS-PAGE and Western blot. Proteins were detected using anti HA, V5 and actin antibodies, and streptavidin HRP. Of note, UBE3A runs as a double band while its catalytically inactive form runs as a single band. - indicates that the cells were transfected with the corresponding empty vector. WT: wild type, CA: catalytically inactive form of UBE3A.