PKC and JNK are involved in DcR3.Fc-induced DC apoptosis. (A) CD14⁺ monocytes (5 × 10⁵) were pretreated with PKC inhibitors for 1 hour before incubation with GM-CSF (200 ng/mL) and IL-4 (10 ng/mL) in the presence of 10 μg/mL Fc-fusion proteins or IgG₁. Apoptotic cells were determined by annexin V–PE/7-AAD staining. *P < .05 compared with control group. PKC inhibitors were safingol (5 μM), rottlerin (5 μM), and Ro-32–0432 (50 nM). (B) In vitro kinase assay of PKC-δ activity in DcR3.Fc-treated DCs. Total cell lysates (300 μg) from different treatments were immunoprecipitated with an antibody against PKC-δ, and in vitro kinase assay was performed in the absence or presence of rottlerin using myelin basic protein (MBP) as substrate. Cell lysates were fractionated on SDS-PAGE, and the phosphorylated MBP was detected by autoradiography. (C) After pretreatment with kinase inhibitors PD98059 (50 μM), SB203580 (30 μM), and SP600125 (20 μM) for 1 hour, apoptotic cells were determined by annexin V–PE/7-AAD staining *P < .05; **P < .01 compared with DcR3.Fc without inhibitor treatment. (D,E) JNK phosphorylation in IgG₁-, DcR3.Fc-, or HBD.Fc-treated DCs in the absence or presence of the PKC-δ inhibitor rottlerin. Cell lysates were fractionated on SDS-PAGE and blotted onto nitrocellulose paper before being probed with anti–phospho-JNK1 mAb. (F) The caspase-8 activity in cell lysates was determined using fluorescent caspase substrates. JNKi indicates JNK inhibitor SP600125 (20 μM). Error bars represent SD of the mean.