Anti p akt1

Cells were lysed for 30 min in Triton buffer (1% Triton X-100, 50 mM Tris-HCl [pH 7.6], 150 mM NaCl, 1% sodium deoxycholate, 0.1% SDS) supplemented with protease and phosphatase inhibitors (1 mM PMSF, 2 mM sodium pyrophosphate, 2 mM sodium betaglycerophosphate, 1 mM sodium fluoride, 1 mM sodium orthovanadate, 10 μg/ml leupeptin, and 10 μg/ml aprotinin). Lysates were cleared by centrifugation at 15,000 × g at 4°C for 15 min, and protein concentrations were determined via the Bradford method. 50 μg of protein were separated by SDS-polyacrylamide gel electrophoresis and transferred onto Immobilon-P membranes. Proteins were detected by using anti-LETM2 (Proteintech, 17180-1-AP), anti-FGFR1 (Abcam cat# ab76464; RRID: AB_1523613), anti-WHSC1L1 (Abcam, ab180500), anti-CDCA7 (Abcam cat# ab69609; RRID: AB_1268064), anti-ERK1/2 (Santa Cruz, sc-514302), anti-p-ERK1/2 (Cell Signaling Technology cat# 4376; RRID: AB_331772), anti-AKT1 (Cell Signaling Technology cat# 2967; RRID: AB_331160), and anti-p-AKT1 (Cell Signaling Technology cat# 9018). Antibody binding was detected using horseradish peroxidase-labeled anti-mouse (Sigma) or anti-rabbit (Cell Signaling) antibodies and chemiluminescence was detected with a LAS4000 device (Fuji). Equal protein loading was confirmed with antibodies against GAPDH (Transgen).