Figure 1

Expression of TWEAK and its receptor, Fn 14 by hCMEC/D3 cells. (A) Steady-state levels of TWEAK, Fn14, and GAPDH mRNAs were assessed by semi-quantitative RT-PCR in cultures of hCMEC/D3, HUVECs, and THP-1 cells. PCR products were analyzed by electrophoresis on a 2% agarose gel containing ethidium bromide and PCR products of the expected sizes were obtained for TWEAK (522 bp), Fn14 (242 bp), and GAPDH (226 bp) indicating expression of all three genes at the mRNA level. (B) Western blot analysis of protein extracts from hCMEC/D3 or MDA-MB-231 cells (positive control) following separation by SDS PAGE and transfer onto nitrocellulose membranes. Membranes were probed with a primary antibody against TWEAK. Note the absence of TWEAK protein expression by the hCMEC/D3 cells as compared with the MDA-MB-231 cells. (C) FACS analysis of membrane bound Fn14 on differentiated hCMEC/D3 and HUVECs using anti-human Fn14 or isotype control phycoerythrin-conjugated antibodies. Note the presence of Fn14 at the plasma membrane of the hCMEC/D3 cells.