Yeast two-hybrid (Y2H) and pulldown assays.
A Y2H. pGADT7-FaeG and pGBKT7-APN were co-expressed in yeast and positive clones were tested for β-galactosidase activity. Samples 1–3: F4ab FaeG-APN; Samples 4–6: F4ac FaeG-APN; Samples 7–9: F4ad FaeG-APN; N: negative control; P: positive control. B GST-pulldown assays. The binding between the recombinant FaeG and APN proteins was studied using the Pierce™ GST Protein Interaction Pull-Down Kit. Western blotting with anti-F4 monoclonal antiserum and anti-APN polyclonal antiserum was used for detection. C Metaperiodate treatment. PVDF membranes were treated with either 0, 10, or 20 mM NaIO4 before using the membranes in Western blots as described in B. Each experiment was repeated three times and representative results are shown.