LMP2A affects activation of Akt and nuclear translocation of Rel proteins in IBL-1 cells. (A) The IBL-1 lymphoma cell line was transfected with siRNA to LMP1 (L1), LMP2A (L2A), or both (L1 + 2A), as well as scrambled (S) as indicated, or mock transfected (−). Proteins were extracted 48 hours after transfection, and probed with antibodies to LMP1 or LMP2A, confirming suppression of the corresponding protein. Actin reprobing was performed to ensure even protein loading. (B) Extracts as in panel A were evaluated for activation of Akt by immunoblotting with an antibody to phospho-Akt substrates, showing that knockdown of LMP2A affects this pathway more significantly than knockdown of LMP1. (C) Nuclear extracts were prepared from cells 48 hours after transfection as in panel A, and probed for the Rel proteins is indicated. Suppression of both LMP1 and LMP2A resulted in significant decrease of nuclear p65 and p50, as well as RelB and p52, showing down-regulation of both the classical and alternative NF-κB pathways. Reprobing with antibodies to histone H2A was performed to confirm similar amounts of nuclear proteins.