Cip2a

T-ALL cell lines were cultured in the presence or absence of 1 μM OP449, 1 μM dovitinib, or combination. Following the indicated drug exposure time, cells were washed in PBS and lysed in 50 μL lysis buffer (9803, Cell Signaling Technology, Boston, MA) supplemented with complete protease inhibitor and phosphatase inhibitor cocktail-2 (Sigma-Aldrich, St. Louis, MO). Equal amounts of protein were fractionated on 4–15% Tris-glycine polyacrylamide gels (Bio-Rad, Hercules, CA), transferred to PVDF membranes and probed with antibodies to SETBP1 (98222) and p-cMYC (78318) from AbCam, San Francisco, CA; c-MYC (764) from Santa Cruz, Dallas, TX; p-AKT (4060), AKT (9272), p-ERK1/2 (9101), ERK1/2 (4695), p-p70S6K (9234), and p70S6K (2708) from Cell Signaling, Danvers, MA; p-Tyr (05-321), actin (C4), and PP2A-C (05-421) from Millipore, Temecula, CA; SET (A302-261a) from Bethyl, Montgomery, TX; CIP2A (a gift from Jukka Westermarck, Finnish Cancer Institute, Turku Centre for Biotechnology, Turku, Finland), p-PP2A-Y307 (1155-1) from Epitomics, Cambridge, MA; and GAPDH (AM4300) from ThermoFisher, Carlsbad, CA.

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).