Phlpp

Protein lysates of cells growing as a monolayer were prepared as described previously [40 (link)]. Protein concentrations were determined by the Bradford assay (Biorad). 30 μg of protein were loaded on a 4–12% Bis-Tris gel (Invitrogen) and blotted onto a nitrocellulose membrane.
Immunohistochemistry of paraffin-embedded sections was performed as described previously [45 (link)]. Antigen-retrieval consisted of microwaving in 0.01 M citrate buffer (pH 6.0) for 10 min. Immunoperoxidase-based detection was performed using the Histostain-Plus 3rd Gen IHC Detection Kit (Invitrogen/Thermo Fisher Scientific) according to manufacturer's recommendations. Cells were analyzed using an Olympus AX70 epifluorescence microscope equipped with a SpotRT digital camera.
Primary antibodies used for immunoblotting and immunohistochemistry were ABL1, CDK2, pTyr (all Santa Cruz), actin (Sigma), pABL1 Y412, pAKT S473, AKT, pCDK2 T160, cleaved caspase 3, pCRKL Y207, CRKL, pKIT Y719, pMAPK p42/44 T202, pPDK1 S241, PDK1, PP2A, pS6K T389, S6K (all Cell Signaling Technologies), CIP2A, PHLPP, SET (all Bethyl Laboratories), cyclin A (Novocastra), KIT (DakoCytomation) and MAPK (Invitrogen/Thermo Fisher Scientific).

Cells were lysed in lysis buffer and proteins (30 μg) were separated on 10–12% SDS/PAGE gel and then transferred onto PVDF membranes (Millipore, Billerica, MA, USA). After blocking membranes, they were incubated with appropriate dilutions of specific primary antibodies against β-actin (from Sigma-Aldrich, St. Louis, MO, USA), Phospho-STAT3 (Tyr705), pAKT(S473), AKT, Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), p44/42 MAPK (Erk1/2) (from Cell Signaling, Danvers, MA, USA), FKBP5, PHLPP (from Bethyl, Montgomery, TX, USA), AR, STAT3 (from Santa Cruz, Dallas, TX, USA), and HIF-2α (from Abcam, Cambridge, MA, USA). The blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies and visualized using the ECL system (Thermo Fisher Scientific, Rochester, NY, USA).