Aiolos l 15

For Western-Blot analysis, SKO-007(J3) cells were pelleted, washed once with cold phosphate-buffered saline, resuspended in lysis buffer [1% Nonidet P-40 (v/v), 10% glycerol, 0.1% SDS, 0.5% Sodium Deoxycholate, 1 mM phenyl-methyl-sulfonyl fluoride (PMSF), 10 mM NaF, 1 mM Na3VO4, complete protease inhibitor mixture (Roche) in PBS] and subsequently incubated 30 min on ice. The lysate was centrifuged at 14000g for 15 min at 4°C and the supernatant was collected as whole cell extract. Protein concentration was determined by the BCA method (Pierce). Thirty to 50 μg of cell extract was run on 12.5% denaturing SDS-polyacrylamide gels. Proteins were then electroblotted onto nitrocellulose membranes (Schleicher&Schuell), stained with Ponceau to verify that similar amounts of proteins had been loaded in each lane, and blocked in 3% milk in TBST buffer. Immunoreactive bands were visualized on the nitrocellulose membranes, using horseradish-peroxidase-coupled goat anti-rabbit or goat anti-mouse immunoglobulins and the ECL detection system (GE Healthcare Amersham), following the manufacturer's instructions. Antibodies against β-actin, IRF4 (H-140), Ikaros (H-100) and Aiolos (L-15) were purchased from Santa Cruz Biotechnology. Antibody against Blimp-1 was purchased from Cell Signaling.

For Western blot analysis, SKO-007(J3) cells were pelleted, washed once with cold phosphate-buffered saline, resuspended in lysis buffer [1% Nonidet P-40 (v/v), 10% glycerol, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM phenyl-methyl-sulfonyl fluoride (PMSF), 10 mM NaF, 1 mM Na3VO4, complete protease inhibitor mixture Roche in PBS], and subsequently incubated 30 min on ice. The lysate was centrifuged at 14,000 g for 15 min at 4 °C, and the supernatant was collected as whole cell extract. Protein concentration was determined by the BCA method Pierce. Thirty to 50 μg of cell extract was run on 10% denaturing SDS-polyacrylamide gels. Proteins were then electroblotted onto nitrocellulose membranes (Schleicher & Schuell), stained with Ponceau to verify that similar amounts of proteins had been loaded in each lane, and blocked in 5% BSA in TBST buffer. Immunoreactive bands were visualized on the nitrocellulose membranes, using horseradish-peroxidase-coupled goat anti-rabbit or goat anti-mouse immunoglobulins and the ECL detection system (GE Healthcare Amersham), following the manufacturer’s instructions. Antibodies against β-actin, IRF4 (H-140), Ikaros (H-100), and Aiolos (L-15) were purchased from Santa Cruz Biotechnology. Antibody against Brd-4 was purchased from Abcam. Antibody against the p85 subunit of PI3 kinase was purchased from Millipore.