Journal: Nature Communications
Article Title: A broadly neutralizing anti-influenza antibody reveals ongoing capacity of haemagglutinin-specific memory B cells to evolve
Figure Lengend Snippet: 3I14 blocks trypsin-mediated HA maturation and pH-dependent conformational changes. ( a ) Trypsin Cleavage Inhibition Assay. 0.4 μg recombinant H3-histidine (H3-BR07) was incubated in the presence of 2.5 μg 3I14 or Fm-6 IgG1, or in the absence of antibody in Tris-HCl buffer at pH 8.0 containing 2 μg ml −1 Trypsin at 37 °C. Trypsin digestion was stopped at several time-points by boiling the sample in a 100 °C water bath. Samples were run on 10% reduced SDS–PAGE and blotted using a HisProbe-HRP Abs. Data represent a representative experiment from three independent experiments. ( b ) 3I14 IgG1 prevented by low-pH triggered conformational rearrangements on the surface-expressed H3-A2/68 and H3-BR07. Upper panels show four various conformations of HA: uncleaved precursor (HA0, left); trypsin in neutral pH cleaved (mature HA, left middle); fusion pH cleaved (mature HA, right middle) and trimeric HA2 (HA2, right). The conformation rearrangements of surface-expressed H3 were detected by FACS staining of 3I14 (solid bars) and the head binding control mAb E730 (open bars). Binding is expressed as the percentage of binding to untreated HA (HA0). For this antibody inhibition assay, H3 was pretreated without mAb, with 3I14, or with control Ab, Fm-6 IgG1 before exposure of the cleaved HAs to pH 4.9. Data represent mean+s.d. of three independent experiments. SDS–PAGE, SDS–polyacrylamide electrophoresis.
Article Snippet: Samples were run on 10% SDS–polyacrylamide electrophoresis gel under reducing conditions and blotted using a HisProbe-horseradish peroxidase and SuperSignal West HisProbe Kit (Pierce Biotechnology, Rockford, IL).
Techniques: Inhibition, Recombinant, Incubation, SDS Page, FACS, Staining, Binding Assay, Electrophoresis