Laemmli buffer

Raji cells were plated at 10⁵ cells ml⁻¹ in 10 cm diameter dishes and dosed with the indicated treatment. Cells were washed with cold PBS and lysed for 10 min in lysis buffer (50 mM Tris–HCl pH 7.4, 1 mM EDTA, 150 mM NaCl, 1% Triton X-100) containing protease inhibitors (Complete, Roche), 1 mM PMSF and phosphatase inhibitors. Samples were clarified by centrifugation at 14 000 × g for 10 min, quantified with Bradford reagent (Bio-rad), denatured by boiling in Laemmli buffer (LI-COR) for 5 min and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis using AnyKD gradient gels (Biorad). Protein was transferred to a PVDF membrane (Millipore) and blocked with Odyssey blocking buffer (LI-COR). Both primary antibodies and appropriate IR-dye conjugated secondary antibodies (LI-COR) were incubated in blocking buffer with 0.2% Tween. Anti-actin was used to control for equal loading and experiments were done in at least two independent biological replicates. Bands were visualized on a LI-COR Odyssey infrared imager.