CD40-dependent EC activation by Jurkat D1.1 (CD154⁺) T cells induces MAPK and SAPK signaling pathways. (A) HUVECs were either left untreated (lane 1) or coincubated with Jurkat D1.1 (CD154⁺) cells for the indicated times (lanes 2-4). Cell extracts (40 μg total protein/sample) were separated by 10% SDS-PAGE, subjected to Western blotting, and reacted with anti–phospho ERK1/2, anti–phospho JNK, or anti–phospho p38 antibodies. (B,C) HUVECs were transfected with siRNA-2 or msiRNA-2 and further stimulated with Jurkat D1.1 (CD154⁺) cells for the indicated times. Western blots were performed as in panel A, and probed with anti-CD40 MoAb (B), or with antibodies detecting either total or phosphorylated ERK1/2, JNK, and p38 isoforms (C). The bottom bands of the panels indicate β-actin protein levels used to normalize for equal loading of the gel lanes. All blots shown are representative from 3 different experiments.