Escherichia coli–expressed recombinant human VEGF-A (hVEGF-A; 165-amino-acid isoform; R&D, Inc., South San Francisco, CA) or E. coli–expressed recombinant rabbit VEGF-A (rVEGF-A; 164-amino-acid isoform with GST tag; Abnova, Walnut, CA) were reduced with 50 mM dithiothreitol and loaded on a 15% SDS-PAGE (Invitrogen, Carlsbad, CA). After completion of electrophoresis, transfer (2 hours) was performed by semidry transfer cell (Bio-Rad, Hercules, CA). Polyvinylidene difluoride (PVDF) membranes were incubated overnight with bevacizumab at a final concentration of 10 μg/mL in blocking buffer (5% skim milk in PBST). After three washes in 0.2% skim milk in PBST, the blots were incubated with peroxidase-conjugated, Fab-specific goat anti–human IgG (ImmunoPure; Pierce, Rockford, IL) for 1 hour at a dilution of 1:10,000 and were developed using enhanced chemiluminescence (ECL Plus Western Blotting Detection System; GE Healthcare Bio-Sciences, Little Chalfont, UK) after 1-minute exposure of the film.