Downstream mediators of receptor signaling remain functional in nonresponsive CLL samples. (A) CLL samples were treated for up to 6 hours with IL-21 and lysates examined by western blot. Induction of PRDM1α, STAT3 Tyr-phosphorylation (pY-STAT3), and MCL1 are shown. Total STAT3 and β-actin were used as loading controls. Representative responsive and nonresponsive CLL samples are shown. (B) Three responsive (R) and nonresponsive (NR) CLL samples were harvested directly after thawing, or after 2 or 20 hours of treatment with and without CpG-ODN. Nuclear extracts were then subjected to an NF-κB p65 subunit ELISA. (C) Induction of IκBα and IκBζ RNA following treatment with CpG-ODN (for up to 15 hours) in 3 R and 4 NR-CLL samples. (D) CLL cells were left untreated or treated with CpG-ODN, PMA (50 ng/mL), or PMA and ionomycin (50 ng/mL and 1 µg/mL, respectively). Cell lysates were then western-blotted and probed for PRDM1α. Representative of (n = 3 each) R- and NR-CLL samples.