Role of ubiquitination in targeting Vpr for autophagy-mediated degradation.

作者：Yuexuan Chen (11424022), Susanne Klute, Anju Bansal (56201), Konstantin M. J. Sparrer, Ruth Serra-Moreno · 发表于：Figshare · 年份：2026 · DOI：10.1371/journal.ppat.1014020.g010 · 研究领域：Molecular biology、Chemistry、Biology、Cell biology

(A) HEK293T cells were transfected with 2,000 ng of Lab-Vpr. 24 h later, DMSO or TAK-243 (200 nM) was added. 44 h later, cells were treated with increasing amounts of Torin2 (5-10 nM). 4 h later, cells were harvested and analyzed by western blot. Membranes were probed for Myc, LC3 and ACTB. The LC3-II/I ratio as well as the Vpr levels relative to DMSO (D on the x axis) are provided over the Torin2 treatment range for both DMSO- and TAK-243-treated cells. Dotted line represents 2-fold threshold. Data correspond to the mean and SEM of 3 independent experiments. (B) HEK293T cells were transfected with Lab-Vpr or the Lab-Vpr K 27 M mutant. 44 h later, cells were treated with DMSO or Torin2 (10 nM). 4 h later, cells were processed for western blot analyses as in panel A. Blots are representative of 3 independent experiments. (C) HEK293T cells were transfected with either Lab-Vpr or TFV-Vpr. 48 h later, cells were harvested, and lysates were immunoprecipitated (IP) for Vpr. The IP samples were analyzed by western blot by probing membranes for ubiquitin and Vpr. The whole cell lysates were analyzed for Vpr and ACTB. Blots are representative of 3 independent experiments. (D) Diagram of SQSTM1 and truncation mutants. (E) HEK293T cells were co-transfected with Lab-Vpr and the mCherry-SQSTM1 constructs. 48 h later, cells were harvested, and lysates were immunoprecipitated (IP) for Vpr. The IP samples were analyzed by western blot by probing membranes for mCherry and Myc. The ...