Figure 2.
Analysis of the binding of CNF1 mutants to Lu/BCAM. (A) amino acid sequence of CNF1 α-helix of interest from amino acid 720 to 733 in comparison to CNFY, * indicate mutated amino acids; (B) dot blot binding studies, different CNF proteins were dotted on a nitrocellulose membrane and incubated with rLu/BCAM for 30 min. After blocking the membrane with skimmed milk, detection of rLu/BCAM was performed by an anti-Lu/BCAM antibody. As a loading control, the membrane was incubated with an anti-GST antibody; and, (C) flow cytometry competition studies, suspension of HeLa cells (3 × 105 cells in 1 mL medium) were preincubated with several unlabeled CNF proteins in different molar ratios, respectively. Then, cells were washed with PBS and incubated with DyLight488-labeled GST-CNF1 (as control cells were incubated with labeled CNF1 without preincubation and set to 100%). Following washing with PBS, cells were subjected to flow cytometry measurements. Bound fluorescence is shown in comparison to the control. Data shown represent three independent experiments + standard deviations. Statistical analyses were performed using two-way ANOVA. ** p < 0.01; *** p < 0.001; **** p < 0.0001.