Fig. 3: UBE3A-mediated ubiquitination targets ABCA1 for proteasomal degradation.

A–C ABCA1 protein level in wild-type (Wt), Ube3a-overexpression (B, Ube3a^OE, n = 3 samples), and Ube3a^−/− (C, n = 4 samples) bone marrow-derived macrophages (BMDMs), normalized to Cy5 levels (loading control). D–G ABCA1 protein level in HEK293T cells in which mUBE3A-Iso2 (E, n = 5 samples), mUBE3A-Iso3 (F, n = 3 samples), or catalytically inactive mUBE3A-Iso2 (mUBE3A-Iso2^C.I., G, n = 6 samples) was expressed. HEK293T cells incubated with equal amounts of PEI served as controls (Ctrl). ABCA1 protein levels are normalized to Cy5 levels (loading control). H Escherichia coli (E. Coli) cells expressing E1, UbcH5 (E2), and with (+) or without (−) ubiquitin (Ub), were transfected with either wtUBE3A or catalytically inactive UBE3A (UBE3A^C817A), in combination with HA:UBE3A, V5:RING1B, or V5:ABCA1 (V5 linked to C-terminal cytoplasmic tail of ABCA1). Cell lysates were analyzed by immunoblotting for HA to visualize UBE3A, or V5 to visualize RING1B or ABCA1. Representative blots of three independent technical replicates are shown. I Immunoprecipitation (IP) of ubiquitin on Wt and Ube3a^−/− Mye⁷²-treated BMDM cell lysates. Whole cell lysates (Input) and final eluate (IP: Ub) were analyzed by immunoblotting for ubiquitin and ABCA1. J IP of ABCA1 on cell lysates of untreated (−) and Mye⁷²-treated Wt BMDMs. Whole cell lysates (input) and final eluate (IP: ABCA1) were analyzed by immunoblotting for UBE3A and ABCA1. Cy5 served as a negative control. Data from A–G are representative of three independent experiments, and all data were shown as mean ± SEM. Statistical significance was determined by a two-sided Student’s t-test. *P < 0.05, **P < 0.01. If p values are not indicated, the result was not significant.