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. Author manuscript; available in PMC: 2020 Jan 8.
Published in final edited form as: Cell Rep. 2019 Dec 24;29(13):4608–4619.e4. doi: 10.1016/j.celrep.2019.11.110

Figure 4. Purified Hhat Reconstituted into Artificial Liposomes Mediates Palmitoyl-CoA Uptake.

Figure 4.

(A–C) The 0.2-μm liposomes reconstituted with or without purified Hhat WT or Hhat H379A were pretreated for 15 min with 10 μM TDI-3410 or 10 μM Etomoxir (A) and incubated for 60 min at room temperature with NBD-palmitoyl-CoA (A; n = 4), [125I] IodoPalmitoyl-CoA (B; n = 2), or [14C] palmitoyl-CoA (C; n = 2), and palmitoyl-CoA uptake was determined as in Figures 1 and 2.

(D) Liposomes reconstituted with purified Hhat WT were pretreated with the indicated concentrations of TDI-3410 and NBD-palmitoyl-CoA uptake was analyzed as in (A); n = 3.

(E) As in (A). After quencher addition, RFU readings were taken every 2 min for 120 min; n = 3.

(F) As in (A). Liposomes were incubated with either NBD-PC or NBD-palmitoyl-CoA; n = 2.

(G) Liposomes reconstituted with purified 157FLAG Hhat were treated with or without trypsin or octyl glucoside (OG), and Hhat was analyzed by SDS-PAGE and western blotting with anti-FLAG antibody.