Figure 3. Binding and anti-migratory activity of AD-01 is dependent on CD44.
(A) Representative graph demonstrating extracts of MDA-MB-231 un-transfected control/NT siRNA transfected cells render increased binding of anti-AD-01 antibody to AD-01 immobilised on CM5 chip surface using the specific binding assay described in Fig. 2. This binding was abrogated in lysates derived from CD44 knockdown cells. Buffer control comprised of HBS buffer with indicated amounts of AD-01. Bar charts demonstrate significant abrogation of this binding in the presence of 10−9 M AD-01 in lysates from cells transfected with CD44 siRNA. Data points show means ± SEM; n = 4. (B) Treatment with AD-01 at 10−9 M results in inhibition of migration in un-transfected and NT siRNA transfected MDA-MB-231 cells. This inhibition of migration was abrogated upon CD44 knockdown using targeted siRNA. Data points show means ± SEM; n = 5. (p-value was determined using one-way ANOVA). (C) Anti-migratory effect of rFKBPL 1500 ng/ml was abrogated in PC3 cells transfected with CD44 targeted siRNA in comparison to those transfected with scrambled siRNA; n = 3.