Blocking activation of the canonical NF-κB pathway through the transfection of IκBα-SR. (A) HA-tagged IκBα-SR (S32A/S36A) or empty pcDNA3 vector was transfected into CLL cells through nucleofection. Twenty-four hours after transfection, the cells were cultured in serum-free medium for 3 hours and then were stimulated with rhBAFF (50 ng/mL) for 30 minutes, and total cell lysates were prepared for immunoblot analysis. MFIR of CLL cells stained for BCMA, TACI, or BR3 was 8.2, 7.0, or 20.8, respectively. Phosphorylation and degradation of IκBα were seen in nontransfected CLL cells and empty vector–transfected CLL cells when these cells were stimulated with rhBAFF. On the other hand, phosphorylation of IκBα was not seen in IκBα-SR–transfected CLL cells. High levels of IκBα were seen in CLL cells transfected with IκBα-SR that were not affected by stimulation with rhBAFF. (B) Samples from each of 8 CLL patients were split and then transfected with an empty control vector or with IκBα-SR. This graph presents the viability of each CLL sample 24 hours after transfection. For each patient sample, the IκBα–SR–transfected CLL cells underwent apoptosis more readily than did control CLL cells transfected with the empty control vector 24 hours after transfection (P < .005; Student paired t test). (C-D) CLL cells were transfected with the empty control vector or with IκBα-SR. Four hours after transfection, the cells were cultured with or without rhBAFF (50 ng/mL) or rhAPRIL (500 ng/mL) for 24 hours. In empty control vector–transfected cells, the viability of CLL cells cultured in medium with rhBAFF or rhAPRIL was significantly higher than that of CLL cells cultured in medium alone (P < .005, P < .05, respectively; Student paired t test). The survival of IκBα–SR transfected cells could not be enhanced by rhBAFF or rhAPRIL.