Under denaturing conditions all forms migrated as expected for gp140. In the lower part of Panel A is the gel obtained by electrophoresis under non-denaturing conditions. In this gel the predominant form of gp140 migrated as dimer, while gp140-GCN4 and gp140-GCN4-L each migrated as trimer with approximate molecular radii of 720 kDa. The gp140L migrated in parallel with gp140, and gp140-L did appear to contain both monomeric and dimeric forms. The size estimates for each of the species were confirmed by Silydianin analytical ultracentrifugation, as shown in Figure 1B. The calculated sizes of each were fully consistent with the oligomeric state predicted by results of electrophoresis. Shown in Figure 2A is specific mAb binding in the presence and absence of sCD4 assessed by immunoprecipitation followed by Western blot detection. We have shown previously that R2gp140 oligomer exhibits the ability to be recognized by CD4i mAbs both with and without CD4 binding, whereas other gp140 strains require CD4 binding for CD4i mAb binding to occur. Figure 2A demonstrates the characteristic binding Diosgenin-glucoside reactivity to a panel of CD4i human mAbs and of a CD4-gp120 epitope complex specific mAb, CG10. As we have previously observed, the binding of CD4 was not required for efficient interaction of any of the CD4i antibodies with R2g140. Similar results were obtained for gp140-GCN4, gp140-GCN-L, gp140-L-Monomer, and gp140-L-Dimer, indicating that CD4i epitopes are exposed on R2gp140 in all of the forms tested here. As expected, CG10 mAb reactivity was completely dependent on CD4 binding to the proteins, since CG10 recognizes the gp120-CD4 complex. The results of mAb binding to the different proteins in ELISA are shown in Figure 2B. The mAbs 2F5 and 4E10 bind to a membrane proximal epitope region in gp41, both neutralize R2 and both bound the different forms of R2 well. The PGT121 and PGT126 mAbs bind to an N-linked glycan in V3 and other variable loop amino acids. They bound similarly to each of the forms of gp140. Both neutralize R2 strain well. The VRC01 and VRC03 mAbs both target the CD4 binding site. VRC01 neutralizes the R2 strain well, while VRC03 neutralizes, but less well.