Fig. 4. UBE4A was recruited by IP6K1 to ubiquitinate apoA-I.

A, Flag-apoA-I and HA-UBE4A were overexpressed together in primary murine hepatocytes. The cells were treated with M132 (10 μM, 4 hours) to block proteasomal activity. Flag-apoA-I was immunoprecipitated and blotted for ubiquitin. B, Flag-apoA-I was overexpressed in primary murine hepatocytes, and UBE4A was deleted by shRNA transduction (the shRNA2 as in panel D). The cells were treated with M132 (10 μM, 4 hours) to block proteasomal activity. Flag-apoA-I was immunoprecipitated and blotted for ubiquitin. C and D, UBE4A was deleted in AML12 murine hepatocytes. (C) Deletion of UBE4A upregulated apoA-I protein levels. Data represent mean±SEM, One-way ANOVA, n=3 independent repeats. (D) The protein levels of apoA-I in the cell culture media were higher in the UBE4A-deleted preparations. Data represent mean±SEM, One-way ANOVA, n=3 independent repeats. E and F, Flag-apoA-I was overexpressed in WT and IP6K1 KO primary murine hepatocytes. (E), Immunoprecipitation of flag-apoA-I co-pulled down less UBE4A in the IP6K1 KOs. Data represent mean±SEM, Student’s t-test, n=3 independent repeats. (F), Immunoprecipitation of UBE4A co-pulled down less flag-apoA-I in the IP6K1 KOs. Data represent mean±SEM, Student’s t-test, n=3 independent repeats. G and H, Flag-apoA-I was overexpressed in primary murine hepatocytes. The cells were treated with IP6K inhibitor SC-919 (1 μM, 24 hours). (G), Immunoprecipitation of flag-apoA-I co-pulled down less UBE4A in the SC-919-treated cells. Data represent mean±SEM, Student’s t-test, n=3 independent repeats. (H), Immunoprecipitation of UBE4A co-pulled down less flag-apoA-I in the SC-919-treated cells. Data represent mean±SEM, Student’s t-test, n=3 independent repeats.