Supersignal west pico ecl detection system

Wild type and mutant RAG1-transfected HEK 293 cells were lysed for 30 min in ice-cold RIPA lysis buffer system (Santa Cruz Biotechnology, USA), and insoluble material was removed by centrifugation (16,000 x g, 10 min, 4°C). 20μg of protein were resolved in 8%SDS-polyacrylamide gel electrophoresis (SDS-PAGE) buffer, electrotransferred onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore), and immunoblotted with anti-RAG1 antibody (H-300) (Santa Cruz Biotechnology Inc; USA). Detection was performed using the SuperSignal West Pico ECL detection system (Thermo Scientific; USA).

EBV-transformed B-cells were lysed for 30 min in ice-cold lysis buffer [20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, 1 % Nonidet P-40, protease inhibitor cocktail (Complete; Roche)] and insoluble material was removed by centrifugation (16,000 x g, 10 min, 4 °C). 20 micrograms of protein was resolved by 8 % SDS-polyacrylamide gel electrophoresis (SDS-PAGE), electrotransferred onto polyvinylidene difluoride membrane (Immobilon-P; Millipore), and immunoblotted with anti-JAK3 antibody (C-21; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-Stat5 antibody (89; BD Biosciences) and anti-GAPDH antibody (Santa Cruz). Detection was performed using the SuperSignal West Pico ECL detection system (Thermo Scientific, Waltham, MA, USA).

Epstein-Barr-virus transformed lymphoblastoid cell line of patient A, patient B and a healthy control were lysed for 30 min in ice-cold RIPA lysis buffer system (Santa Cruz Biotechnology, USA), and insoluble material was removed by centrifugation (16,000 × g, 10 min, 4°C). Twenty Microgram of protein were resolved in either 8%SDS-polyacrylamide gel electrophoresis (SDS-PAGE) or in 4% Native-polyacrylamide gel electrophoresis. Samples were subsequently electro transferred onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore), and immunoblotted with anti-IgM antibody (2C12-3) (Santa Cruz Biotechnology Inc; USA). Detection was performed using the SuperSignal West Pico ECL detection system (Thermo Scientific; USA).