FKBP51 directs autophagic pathways.

<p>(A) FKBP51 interacts with Beclin1, Akt, and PHLPP. HEK cells were transfected with vector control (lanes 1 and 3) or a FLAG-tagged FKBP51-expressing plasmid (lanes 2 and 4) and lysed 72 h later. After immunoprecipitation (IP) of protein complexes using a FLAG antibody, input (lanes 1 and 2) and (co)precipitated (lanes 3 and 4) proteins were visualized by Western blotting. (B) Change of total Beclin1, pAkt^S473, and pAkt^T308 upon expression of FKBP51. Representative Western blots on the right. FKBP51 was detected by an antibody directed against its FLAG tag. (C) FKBP51 interacts preferentially with dephosphorylated Akt and promotes Akt dephosphorylation. Graphs display quantification of pBeclin1 (S234 and S295) and Beclin1-Akt/pAkt^S473 interaction after Beclin1 immunoprecipitation from HEK cells transfected with FKBP51/52 (details in <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.1001755#pmed.1001755.s002" target="_blank">Figure S2</a>). (D) Comparison of the endogenous protein levels of effects of Beclin1, LC3B-II/I, Atg12, and pAkt^S473 levels in wild-type MEFs, 51KO MEFs, and 51KO MEFs transfected with FKBP51 or vector control. Graphs show the relative expression, with the levels of wild-type cells transfected with vector control set to 1 (dashed line). Representative Western blots on the right. (E) Quantification of Beclin1-Akt interaction after Beclin1 immunoprecipitation in brain extracts from wild-type and 51KO mice. Graph represents the results from five independent experiments. Representative Western blots on the right. (F) FKBP51 enhances autophagic flux. Cortical rat astrocytes were transfected with FKBP51 or vector and treated with bafilomycin A₁ (BafA1) as indicated, and the levels of LCB3-II/I were determined. (G) Cellular effects of FKBP51 on autophagic markers require Akt1 and/or Akt2. The protein levels of Beclin1 and LC3B-II/I were evaluated in wild-type MEFs, Akt1/2KO MEFs, and Akt1/2KO MEFs transfected with Akt1- and Akt2-expressing plasmids. Graphs display the relative expression of three different experiments; expression in wild-type vector-transfected MEFs was set to 1 (dashed line). Representative Western blots on the right. *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001. See <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.1001755#pmed.1001755.s013" target="_blank">Table S1</a> for statistical details. ect., ectopic; genot., genotype; KO, 51KO; WT, wild-type.