Modification

transfection of Smc3 with ATPase A point mutation

ATPase Walker A motif of Smc3 (one of the Condensin SMC complex proteins) is distrupted with transduction. Mouse splenic B lymphocytes were preactivated overnight in the presence of 0.5 mg/mL of aCD180 (RP105) antibody. At 24 hr cells were transduced with Smc3 (K38A) vectors (pMy-Smc3_biotag-T2A-mOrange) and pMy-BirA-T2A- eGFP by centrifugation for 90 min at 2,500 rpm, at 32C. B cell media was supplemented with 50 mg/mL of LPS, 2.5 ng/mL of IL-4, and 0.5 mg/mL of aCD180. At 32 hr, cells were diluted to 0.1x106 cells per mL. Seventy-two hours after first infection, B cells were harvested and GFP/mOrange double positives were cell sorted using a BD FACSAria III (Becton Dickinson).