Fig. 5.
Immunoblots of proteins bound by GST-PFD and eluted with GSH. Aliquots of gD260t (40 ng each) alone (reaction mixture 4) or mixed with soluble HVEMt (reaction mixture 2), nectin 1-Fc (reaction mixture 3), or nectin 3-Fc (reaction mixture 1) for 2 h at 4°C were reacted with GSH-beads preloaded with GST-PFD. Replicate aliquots of the bound proteins, eluted with GSH, were separated by SDS/PAGE and blotted, and the blots were developed with mAb H170 to gD (A), anti-His antibody to HVEMt (B), CK6 mAb to nectin 1-Ig (C), or anti-human IgG to nectin 3-Ig (data not shown). Input controls (C) contained the indicated proteins (gD260t in A, HVEMt in B, and nectin 1-Fc in C) and were loaded in SDS/PAGE for control of migration position and immunoreactivity. It can be seen that, when gD260t was premixed to soluble HVEMt (reaction mixture 2) or nectin 1-Ig (reaction mixture 3), neither gD260t (A) nor the receptors (B and C) were present in the GSH-eluted proteins.