Expression of activated JNK and cJun proteins and AP-1 DNA binding activity in NPM-ALK⁺ ALCL cells. (A) Two NPM-ALK⁺ ALCL cell lines, Karpas 299 and SU-DHL1, were examined by immunoblot analysis using antibodies specific for JNK, p-JNK, cJun, and its phosphorylated forms, ^Ser73p-cJun and ^Ser63p-cJun. JNK1 and JNK2 are expressed as isoforms of 46 kD and 54 kD, respectively, generated by alternate splicing. Both cell lines were found to express high levels of JNK and cJun, as well as their phosphorylated forms, with ^Ser73p-cJun being more prominent. Two Hodgkin lymphoma cell lines, L-428 and L-1236, served as positive controls and REH cells, derived from a patient with pre-B-ALL, served as a negative control, respectively, for expression of cJun and p-cJun (data not shown). (B) Immunochistochemistry was performed on formalin-fixed, paraffin-embedded Karpas 299 and SU-DHL1 cell blocks using the same antibodies for detection of cJun and their phosphorylated/activated forms. Both NPM-ALK⁺ ALCL cells revealed nuclear staining for cJun, ^Ser73p-cJun, and ^Ser63p-cJun. (C) AP-1 DNA binding activity was assessed in SU-DHL1 and Jurkat by EMSA using the double-stranded consensus oligonucleotides with AP-1–specific binding. Super shift of protein–DNA complex (arrow) was detected in SU-DHL1 cells when nuclear extracts were preincubated with (+) antibody against cJun, indicating the AP-1 complex in ALK⁺ ALCL cells contains cJun. AP-1 DNA binding activity was undetectable in Jurkat cells that served as a negative control in this experiment. Free DNA is shown in the bottom of the gel.