P foxo3 t32

Prepared IP samples and corresponding whole cell lysates were fractionated by SDS-PAGE and transferred to a nitrocellulose membrane according to the manufacturer (BioRad). Membranes were blocked with 5% BSA in TBS for 30 min and incubated with antibodies against FOXO3 (1:1000; Abcam), P-FOXO3 T32 (1:1000; Cell Signaling), PRDX1 (1:4000; Abcam), PRDX-SO3 (1:500; Abcam), 14-3-3 (1:1000; Cell Signaling), or actin (1:1000; Oncogene) overnight at 4°C. Membranes were washed four times for 5 min in TBST (0.05% Tween-20) and visualized by IR or chemiluminescent detection. For IR processing, membranes were incubated with a 1:15,000 dilution of anti-goat, anti-rabbit, or anti-mouse IRDye (LI-COR) for 30 min at 25°C. Blots were washed with TBST three times and with TBS once, and imaged on an Odyssey (LI-COR) imager. Membranes processed by chemiluminescence were incubated in a 1:10,000 dilution of HRP-conjugated anti-mouse or anti-rabbit antibodies for 1 h at 25°C. Blots were washed four times with TBST for 5 min and exposed to ECL for 1 min.