Figure 5.

Detection of LBP/p40 by mAb 15308 and expression of exogenous LBP/p40 on the surface of apoptotic cells. (a) Exogenous expression of the LBP/p40 cDNA (pcDNA3.1D/LBP/p40-V5-His) in lysates from transiently transfected HEK293T cells (lane: p40) and detection of 40 and 50–70 kDa species by anti-V5 antibody and by mAb 15308 but not by isotype control antibody (γ3) or anti-actin antibody. Lysates from mock-transfected cells are shown for comparison (lane: M). (b) Exogenous expression of the NEDD5 cDNA (pcDNA3.1D/NEDD5-V5-His) in lysates from transiently transfected HEK293T cells (lane: N5) and detection by anti-V5 antibody but not by mAb 15308. NEDD5 was not detected in mock-transfected cells (lane: M) and mAb 15308 detected endogenous 40 kDa bands in lysates from both NEDD5- and mock-transfected cells. Note that the differences in signal intensity between anti-V5 and mAb 15308 can be explained by the exquisitely high affinity of anti-V5 for its epitope. (c) Immunofluorescence microscopic analysis of MCF-7 cells transiently transfected with pcDNA3.1D/LBP/p40-V5-His and double-stained with anti-V5 followed by Alexa Fluor-488-tagged secondary antibody (green, left panel), and mAb 15308 followed by Alexa Fluor 568-tagged secondary antibody (red, centre panel). Right panel shows overlay of red/green fluorescence and arrows indicate an example of an area where coincident fluorescence was observed. (d) Surface exposure of exogenous V5-tagged LBP/p40 stably expressed in MCF-7 cells. Confocal microscopy of MCF-7/V5-LBP/p40 transfectants after 48 h etoposide treatment. Exogenous LBP/p40 was detected using anti-V5 and secondary antibody tagged with Alexa Fluor-488 (green). Plasma membrane integrity was monitored by PI (red) exclusion. Z-sections of rare cells revealed the presence of the V5 epitope on surface blebs with plasma membranes intact (arrows, upper panels) as opposed to membrane compromised cells (arrows, lower panels), which stained green throughout the cell and were positive for PI