Activation of JNK, NF-κB, and c-Jun phosphorylation by hCD40-P227A versus WT hCD40 signaling. (A) CH12.hCD40WT (hCD40WT.2) and CH12.P227A (P227A.1, P227A.2) cells were stimulated for the indicated times with medium (BCM), uninfected Hi5 (Hi5), or Hi5-hCD154. Total cell lysates were resolved by SDS-PAGE and immunoblotted for phosphorylated JNK, followed by total JNK as a loading control. Data are representative of 3 independent experiments with multiple clones tested. Results for P227A.2 are not included in the graph because its lower background produces a different scale. (B) CH12.hCD40WT (hCD40WT.1, hCD40WT.2) and CH12.P227A (P227A.1, P227A.2) cells were stimulated for the indicated times with media (BCM), uninfected Hi5 (Hi5), or Hi5-hCD154. Total cell lysates were analyzed via SDS-PAGE and immunoblotted for phosphorylated c-Jun (Ser63), followed by actin as a loading control. Results were quantified as described in “Immunoblots,” and are representative of 4 independent experiments. (C) M12.hCD40WT (hCD40WT.1, hCD40WT.2) and M12.P227A cells (P227A.1, P227A.2) were stimulated and total cell lysates prepared. Samples were immunoblotted for phosphorylated c-Jun (Ser73) followed by actin as a loading control. Results are representative of 5 experiments.