Figure 7. BAG3 reroutes PTTG1 to autophagy for degradation. (A) HEK293T cells were either left untreated or treated with 10 mM 3-methyladenine and 200 nM wortmannin (3-MA+wort.) or 100 nM bortezomib (bort.) overnight. (B) HEK293T cells were transfected with an empty vector, His-BAG3, or His-BAG3-BAGΔ-encoding vectors. (A and B) Expression levels of PTTG1 were measured using a specific antibody. Average levels of PTTG1 are reported (**P < 0.001 compared with untreated cells; n = 4 independent samples ± sem and *P < 0.01 compared with empty vector; n = 3 independent samples ± sem). (C) HEK293T cells overexpressing empty vector or His-BAG3 were either left untreated or treated overnight with 200 nM wortmannin and 10 mM 3-methyladenine (wort.+3-MA) or with 100 nM bortezomib (bort.) prior to extraction of total proteins. Average levels of PTTG1 are reported (**P < 0.001 compared with empty vector; n = 5 independent samples ± sem). (D and E) PTTG1 partially colocalizes with myc-MAP1LC3B and LAMP2 in cells overexpressing BAG3. (D) HEK293T cells were transfected with a myc-MAP1LC3B-encoding vector and either an empty vector or His-BAG3. Twenty-four hours post-transfection cells were treated with bafilomycin A₁ (100 nM) for 4 h and fixed with formaldehyde; subcellular distribution of myc-MAP1LC3B and endogenous PTTG1 were investigated by immunofluorescence. (E) HEK293T cells were transfected with His-tagged FL or BAGΔ BAG3-encoding vectors. Twenty-four hours post-transfection cells were treated with bafilomycin A₁ for 4 h and fixed with formaldehyde; subcellular distribution of endogenous LAMP2 and PTTG1 were investigated by immunofluorescence. (F) HEK293T cells were transfected with a scrambled siRNA or with a BAG3 and BAG1-specific siRNA. PTTG1 expression levels were measured 72 h post-transfection. Average levels of PTTG1 are reported (**P < 0.001 compared with scrambled siRNA; n = 3 independent samples ± sem).