The total number of cells was then determined using a hematocytometer. The cell suspension was pelleted and lysed in buffer containing 1% cymal-5, 150 mM NaCl, 20 mM Tris-HCl, 2 mM EDTA, 10% glycerol, and complete protease Orientin inhibitor cocktail tablet. 1.5 mg amounts of the different versions of purified R2 gp140 were pre-incubated with or without 1.5 mg of sCD4 for at least 2 hr at 4uC. Then, 16106 Cf2ThCCR5 cells in 700 ml lysis buffer were added to the gp140-CD4 complex or gp140 without CD4 as control. Another reaction with same the volume and amount of Cf2Th cells only was used as control. The reactions were incubated at 4uC for overnight. The next day, 1 mg of 1D4 was added to each reaction and the reaction was incubated for another 4 hrs followed by Protein G Sepharose precipitation for an additional 2 h at 4uC. The precipitated complexes were washed five times with lysis buffer, 5 mins each time at 4uC, and then Brusatol resuspended in SDS-PAGE sample buffer and processed for Western blotting, as above. The blots were then probed for the presence of gp140, CCR5, and CD4 separately using polyclonal rabbit anti gp140 antiserum, polyclonal goat anti-CCR5 CKR5 antibody, and a polyclonal antihuCD4 affinity purified Goat IgG. Animal studies were performed at Spring Valley Laboratories, Sykesville, MD, in accord with applicable ALAAC requirements for animal care and with approval of the Spring Valley Laboratories Institutional Animal Care and Use Committee, SVL-045. Immunizations were carried out in NZW rabbits. Each construct was studied in a group of five rabbits. All groups of rabbits were given 30 mcg gp140 in 0.5 ml of adjuvant on weeks 0, 4, 8, and 26, and on months 8 and 10. The controls and the group receiving gp140-GCN4-L were given a final dose at month 12. In the previous study by Zhang et al, R2 gp140 was administered in AS02A adjuvant. Based on results obtained in other studies since that study was initiated, a decision was made to use the adjuvant AS01B in this study. AS02A and AS01B contain the same key active ingredients formulated differently.