Fig. 2. FKBP51 associates with SKP2 that regulates BECN1 stability.

a HEK293 cells were transfected with plasmids expressing the indicated E3 ligases and USPs; after 72 h, cells were either harvested for immunoprecipitation of FKBP51. b BECN1 associates with SKP2, but not with USP18 or USP36. HEK293 cells were transfected with vectors expressing tagged SKP2, USP18 and USP36; after 72 h, cells were harvested for immune-precipitation with either control IgG or anti BECN1 antibody. c, d SKP2 affects BECN1 stability. HEK293 cells were transfected with plasmids expressing SKP2 or TRAF6 and exposed to MG132 (10 µM, 2 h) where indicated and to cycloheximide (CHX, 30 µg mL⁻¹) after 72 h for the durations indicated to monitor the decay of BECN1. e, f The conditions of c,d were also used in the pulse-chase assay, performed as in Fig. 1e, f, to determine BECN1 stability. g, h Cellular SKP2 levels determine BECN1 levels. SKP2 was either down-regulated by using siRNA (g) or up-regulated by transient transfection (h) and the levels of the indicated proteins were measured by western blotting (P27 served as control). Quantifications for g and h are provided in Supplementary Fig. 1c, d. i Knock-down of SKP2 by siRNA decreases BECN1 ubiquitination (assay as in Fig. 1b). j–m Stability assays were performed like in c–f to determine the effect of SKP2-targeting siRNA on BECN1 stability. WCE = whole cell extract. In all panels, error bars denote the standard error of the mean, derived from n = 3 biologically independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (two-way ANOVAs, details in Supplementary Table 1). Source data and blot collections are provided as a Source Data file.