Prdx so3

Prepared, immunoprecipitated (IP) samples and corresponding whole cell lysates were resolved by SDS-PAGE and transferred to a nitrocellulose membrane according to the manufacturer’s (BioRad) instructions. Membranes were blocked with 5% BSA in TBS for 30 min, and incubated with primary antibodies against JNK1/2 (1:1000)(Invitrogen), P-SAPK/JNK (1:1000) (Cell Signaling), P-c-jun (1:1000) (Cell Signaling), P-ATF2(1:1000) (Cell Signaling), PRDX1 (1:4000)(Abcam), PRDX-SO3 (1:1000)(Abcam), and actin (1:1000) (Oncogene), overnight at 4 °C. Membranes were washed four times for 5 min each in TBST (0.05% Tween-20) and visualized by IR or chemiluminescent detection. For IR processing, membranes were incubated with a 1:15000 dilution of anti-goat, anti-rabbit, or anti-mouse IRDye (LI-COR), for 30 min at 25 °C. Blots were washed three times with TBST, once with TBS, and imaged on an Odyssey (LI-COR) imager. Membranes processed by chemiluminescence were incubated in a 1:10000 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.