Figure 6

(A) Binding and oligomerization of Rv1694 on rRBC membrane. Various amounts of purified Rv1694 were incubated with 2% rRBC for 30 min and the resultant blot was immuno probed with anti-6-histidine-antibody. Lanes indicated with RBC, Sup and numbers respectively indicate rRBC membrane, purified Rv1694, supernatant of rRBC, rRBC incubated with 5.0 μg, 10.0 μg, 20.0 μg and 30.0 μg Rv1694 respectively. (B) Oligomerization of Rv1694 on RBC membrane in the absence of reducing agents: Oligomerization of Rv1694 (10 μmug) on rRBC (2%) was carried out as described in methods section. The samples were washed and dissolved in 4% SDS and 5× laemmli sample buffer without β-mercaptoethanol and immuno probed with anti-6-histidine-antibody. Lane 1. Rv1694 purified protein; Lanes 2-5 show the oligomers of Rv1694 on the RBC membrane at indicated temperatures (°C). (C) Binding and oligomerization of Rv1694 protein on the Phagosomal membrane: Aliquots were taken out at indicated time points from the Rv1694 incubated phagosomal preparations, electrophoresed on 8% SDS-PAGE (non-reducing), immuno probed with anti-6-histidine-antibody. Lane 1: Phagosomal preparation alone; Lane 2: Unboiled Rv1694; Lanes 3 to 5: Unboiled samples of phagosomal preparations and Rv1694 incubated for 10.0, 30.0, and 60.0 min respectively. Lane 6: Boiled Rv1694 protein; Lane 7: Boiled sample of lane 5. Labels viz., M, O3, O5, O6 and O7 indicate monomers, trimers, pentamers, hexamers and heptamers respectively. The blot was stripped and re-probed with anti-LAMP-1 antibody to ascertain the phagosomal preparation. The data shown is one of the three independent experiments.