Anti p flt3

Cultured cells were lysed in lysis buffer containing 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitor cocktail (Roche Applied Science, Basel, Switzerland), and PhosStop^TM phosphatase inhibitor mixture (Roche Applied Science). Protein concentrations were determined using the Quick Start^TM Bradford Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). Lysates containing equal amounts of protein were subjected to SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Target proteins on the membranes were detected with specific primary antibodies and matching horseradish peroxidase-conjugated secondary antibodies and visualized using Western Lightning^® Plus-ECL (Perkin Elmer, Waltham, MA, USA) and a LAS 4000 image reader (Fujifilm, Tokyo, Japan). The primary antibodies used were as follows: anti-FLT3, anti-MEK1/2, anti-p-MEK1/2, and anti-p53 antibodies (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-p-FLT3, anti-ERK1/2, anti-p-ERK1/2, anti-Akt, anti-p-Akt, anti-p-STAT5, and anti-β-actin antibodies (Cell Signaling Technology, Beverly, MA, USA); anti-CREB and anti-p-CREB antibodies (Epitomics, Burlingame, CA, USA); anti-Mcl-1 and anti-GAPDH antibodies (GeneTex, San Antonio, TX, USA).