Fig. 4

Biosensor strategy to determine NS5A AH binding receptor. QCM-D biosensing detected the interaction of AH peptide with model and Huh7 cell-derived membranes. Changes in resonance frequency of oscillation and energy dissipation were monitored. Binding of AH peptide to model membranes. a AH peptide binds to planar lipid bilayers. At 10 min, lipid vesicles were added to a silicon oxide substrate (arrow 1). The vesicles fused and ruptured to form a planar bilayer. After a buffer wash at 30 min, AH peptide was injected at 40 min (arrow 3). In order to confirm specific binding of the peptide, an additional wash with the same buffer was performed (arrow 4). b NH peptide does not bind to planar lipid bilayers. Identical experiments to those in a were performed, but no binding of the control NH peptide to the planar lipid bilayer was detected. Binding of AH peptide to cell-derived membranes. c AH peptide binds to Huh7-derived membranes. At 10 min, Huh7-derived microsomes were injected (arrow 1). After buffer washes at 70 min and 80 min (arrows 2 and 3, respectively), AH peptide was injected at 90 min (arrow 4). In order to confirm specific binding of the peptide, an additional wash with the same buffer was performed (arrow 5). d NH peptide does not bind to cell-derived membranes. Identical experiments to those in Fig. 6c were performed, but no binding of the control NH peptide to Huh7-derived membranes was detected. Biochemical strategy to confirm proteinaceous receptor preference. e Trypsin treatment does not affect the binding of TAT-derived peptide, which binds membranes independently of a protein receptor. AH peptide or TAT-derived peptide was added (arrow 1) to trypsin-treated Huh7 membranes deposited on silicon oxide, as in panel a, followed by a buffer wash 20 min later (arrow 2). f Mass changes associated with binding of the AH and TAT-derived peptides to the trypsin-treated Huh7-derived membranes of panel c, as calculated using the Sauerbrey equation. QCM-D analysis of monoclonal antibody FG6 binding to its ER membrane receptor PTP1B and sensitivity of binding to prior trypsin treatment of the membranes. g Binding of monoclonal antibody FG6 to its ER membrane receptor PTP1B contained in Huh7-derived membranes. At 10 min, Huh7-derived microsomes were injected (arrow 1). After buffer washes at 70 min and 80 min (arrows 2 and 3, respectively), monoclonal antibody FG6 was injected at 90 min (arrow 4). To ensure that the antibody was indeed bound to its membrane receptor, the membranes were washed again with PBS buffer (arrows 5 and 6). h PTP1B does not bind to trypsin-treated Huh7-derived membranes. At 10 min, Huh7-derived membranes were injected (arrow 1), followed by washing twice with PBS buffer (arrows 2 and 3, respectively). Then, trypsin was applied to cleave proteinaceous components (arrow 4), followed by thorough washes with buffer. After the washing steps were repeated four times (arrow 5), the anti-human PTP1B (FG6) antibody was injected (arrow 6) to examine its ability to bind to the trypsin-treated Huh7-derived membrane, followed by additional washing steps (arrows 7 and 8, respectively). Figure is adapted and modified from Ref. [80]