Fig 3.

Characterization of BNC displaying EC-Fc. (A) Schematic representation of the multivalent display of EC-Fc on ZZ-BNC (EC-Fc/BNC) and Fc on ZZ-BNC (Fc/BNC). (B) The solubility of BNC-displaying Fc/BNC and IgG/BNC was evaluated with FITC-labelled BNC. Residual fluorescence in supernatant was measured in varying molecular ratio of Fc or human IgG to ZZ-BNC. The intensity from the FITC-labelled ZZ-BNC without Fc was calculated as 1 in each graph. (C) Western blot analysis of Fc in the supernatant obtained in (B). Fc/BNC in the supernatant was immunoprecipitated with anti-HBsAg antibody conjugated to micro beads and was subjected to Western blotting. The Fc on the blot was detected with anti-human IgG. The bands were densitometrically analysed with ImageJ and relative intensity of each lane was plotted. (D–F) Assessment of internalization of EC-Fc/BNC in SK-BR-3 cells through Western blot. (D) EC-Fc/BNC (40 nM/2 nM) or Fc/BNC (40 nM/2 nM) was incubated with SK-BR-3 cells for 5 hrs at 4°C and 37°C. (E) SK-BR-3 cells were treated with various concentration of EC-Fc/BNC from 1 to 10 nM. Fc/BNC in 10 nM was taken as control. (F) SK-BR-3 cells were treated with 2 nM EC-Fc/BNC at various time periods. Simultaneously 2 nM Fc/BNC was taken as control. (D–E) After the incubation the cells were trypsinised and lysed followed by immunoprecipitation with anti-HBsAg antibody conjugated to micro beads. The precipitates were immunoblotted and were detected with anti–pre-S1 antibody. The bands of BNC were densitometrically analysed by ImageJ and plotted into each graph to evaluate amount endocytosed. (G, H) Confocal microscopic observation of SK-BR-3 cells treated with EC-Fc/BNC or Fc/BNC. Cells were incubated for various time periods (G) and for 4 hrs (H). The RITC-labelled ZZ-BNC was used and the cells were fixed and permeabilized. EC-Fc or Fc were detected with anti-human IgG labelled with FITC (G) and ErbB2 was detected with sc-08 antibody followed by rabbit anti-mouse IgG Alexa 488 (H). Bars, 10 μm.