Anti blimp 1

One hundred-milligram tissue samples of kidneys, liver, spleen and lymph nodes from mice were homogenized in 1 ml homogenization buffer including 50 µg/ml phenylmethylsulfonyl fluoride (PMSF) and 5 µg/ml leupeptin. Cells were frozen at −20 °C for 20 min, followed by high-speed centrifugation (12000 rpm, 20 min) at 4 °C. The supernatant was collected using an EP tube and stored at −80 °C. After quantification of protein concentrations in the supernatant, the lysates were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA). The membrane was blocked with 2% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 for 2 h and probed with rabbit anti-Blimp-1 (1:200), XBP-1 (1:100), C-myc (1:400) (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-GAPDH monoclonal antibody (1:2000, Sigma, St. Louis, MO), respectively, at 4 °C overnight. After washing, the bound antibodies were detected by peroxidase-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies and visualized using an enhanced chemiluminescence system (Santa Cruz Biotechnology). Protein expression level was determined by analyzing the optical density value obtained from Quantity One software (Bio-Rad, Hercules, CA) and was calculated from triplicate samples after normalization to GAPDH levels.