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Figure 3 | BMC Biology

Figure 3

From: Spartin activates atrophin-1-interacting protein 4 (AIP4) E3 ubiquitin ligase and promotes ubiquitination of adipophilin on lipid droplets

Figure 3

Spartin increases activity of atrophin-1-interacting protein 4 (AIP4) and promotes ubiquitination of adipophilin. (a) HeLa cells were cotransfected with Myc-wild-type spartin together with hemagglutinin (HA)-ubiquitin and an empty vector (lane 1), or Flag-AIP4 (lane 2), or Myc-spartin PY/AA with HA-ubiquitin and Flag-AIP4 (lane 3). Cell lysates were boiled in sodium dodecyl sulfate (SDS) before immunoprecipitation (IP) with anti-Flag antibodies. Levels of HA-ubiquitin and Flag-AIP4 in immunoprecipitates and total cell lysates (TCL) were analyzed by immunoblotting. (b) Levels of in vitro ubiquitination assay of glutathione S-transferase (GST)-AIP4ΔC2 alone (lane 2), with spartin155-367 (lane 3), or mutant spartin 155-367PY/AA (lane 4) determined by immunoblot with anti-ubiquitin antibodies (upper panel). All reactions (except lane 1) were carried out in the presence of E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, ATP, and ubiquitin. Coomassie blue stained gel (bottom panel). (c) In vivo ubiquitination assay of HA-adipophilin, also known as adipose differentiation-related protein (ADRP) in HeLa cells transfected with either HA-empty vector or HA-ADRP, and control or spartin small interfering RNA (siRNA) and Flag-ubiquitin and treated with 300 μM of oleic acid. Cell lysates were immunoprecipitated with anti-HA antibodies and immunoblotted with anti-Flag or anti-HA antibodies.

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