Fig. 9.

Ensa impedes the Cullin-dependent degradation of Treslin by preventing its dephosphorylation by PP2A. a Parental HeLa cells were treated with siSC or siEnsa and subsequently incubated for 6 h without (−) MG132, for 2, 4 or 6 h with MG132 (10 µM) or for 8 h with MLN4924 (500 nM) and subsequently recovered for western blot analysis. β-Tubulin levels were used as a loading control. b As for a, except that U2OS cells were used. c U2OS cells stably expressing Treslin cDNA were transfected with siSC or siEnsa and subsequently incubated with (2 h) or without (−) MG132 (10 µM) and Treslin amount checked by western blot. d HeLa cells overexpressing a Flag-tagged wild-type (WT) Treslin or the phosphomimetic mutant of this protein T968E/S1000E (TESE) were incubated with cycloheximide for the indicated periods of time and submitted to western blot to measure ectopic Treslin levels. e As for d, except that U2OS instead of HeLa cells were used. f HeLa and U2OS cells were co-transfected with Flag-tagged wild type or the T968/S1000E Treslin mutant and treated with siSC or siEnsa and used for western blot to measure ectopic Treslin levels g HeLa cells were transfected with scramble (siSC) and Ensa (siEnsa) siRNAs and 24 h later incubated (+) or not (−) with the PP2A inhibitor okadaic acid (OA; 5 nM). Cells were recovered 24 h later and used for western blot. h As for g, except that U2OS instead of HeLa cells were used. i FACs analysis for HeLa cells in g is shown