Phosphorylation of JNK in human neutrophils, undifferentiated HL-60 cells, and ATRA-differentiated HL-60 cells stimulated by G-CSF, GM-CSF, or TNF. Cells were stimulated with G-CSF (50 ng/mL), GM-CSF (5 ng/mL), or TNF (100 U/mL) for 10 minutes at 37°C. Phosphorylation of JNK was analyzed by immunoblotting using antibody against phosphorylated form of JNK (upper panel). The existence of JNK protein was analyzed by immunoblotting using antibody, which is produced by immunizing rabbits with a full-length JNK2 fusion protein and recognizes both phosphorylated and unphosphorylated forms of JNK (lower panel). Total extracts from human embryonic kidney 293 cells prepared with UV light treatment were used as phosphorylation-positive controls. The cell lysates equivalent to 4.7 × 10⁶ neutrophils, 1.9 × 10⁶undifferentiated HL-60 cells, or 1.9 × 10⁶ differentiated HL-60 cells were loaded onto each lane. The results shown are representative of four independent experiments.