P crkl y207

General biochemicals and reagents were obtained from Sigma Aldridge (Sigma Aldridge, Poole, UK) unless otherwise specified. Fluorochrome-conjugated antibodies recognising the following antigens were employed: CD19, CD5, CD23, CD14, CD3, CD4 all from (BD Pharmingen, Oxford, UK). Unconjugated antibodies recognised: CD29 (clone P5D2 Abcam, Cambridge, UK), CRKL (ab32018 Abcam), P-CRKL (Y207) ( #3181 Cell Signaling Technology, New England Biolabs, Hitchin, UK), ABL (ABL clone 8E9 Abcam), ARG (181B11 Abcam). Secondary antibodies used for immunofluorescence were (Goat anti-mouse FITC Ab5999 and goat anti-mouse tr ab6003 Abcam). Phalloidin probes for F-actin were nitrobenzoxadiazole (NBD)-pallacidin (Invitrogen) and Texas Red phalloidin (Invitrogen). Other agents were CXCL12 (Peprotech EC Ltd, London, UK); Y27632 (Tocris Bioscience, Bristol, UK); and imatinib (LKT Laboratories, Alexis Corporation, Lausen, Switzerland). Annexin V FITC Staining kit (BD Pharmingen) was used with manufacturers protocols to determine apoptotic cell death. Immunoperoxidase staining used standard reagents and protocols (DAKO UK Ltd, Ely, UK).

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).