Ab137718

LCLs for the family were grown as described above (RNA extraction and cDNA synthesis), as well as HeLa cells which were used as a positive control for each antibody. LCLs were harvested by centrifugation at 900 rpm for 5 min and cell pellets washed in phosphate-buffered saline. All cells were lysed in RIPA buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 150 mM NaCl, 1 mM EDTA, Roche complete protease inhibitor). Post-nuclear supernatants were heat-denatured in reducing Lithium dodecyl sulfate loading dye and run on a 4–12% Bis-Tris gel (Invitrogen) under reducing conditions (30 μg total protein loaded per lane). Proteins were transferred onto Polyvinylidene fluo-ride membrane, and Western blotting was performed according to standard protocols using a monoclonal antibody raised against the C-terminus of anti-CD38 (ab108403, Abcam, Cambridge, UK) or a polyclonal antibody raised against amino acids 1–146 of anti-bone marrow stromal cell antigen 1 (ab137718, Abcam), followed by appropriate secondary antibodies (Bio-Rad, Hemel Hempstead, UK). Immunoreactive bands were visualized using ECL Plus Western blot detection reagent (GE Healthcare, Little Chalfont, UK).