Figure 3.

Bevacizumab interacted with, induced phosphorylation of, and upregulated abundance of FcγRI in vivo. (a) Wild-type mouse corneas that had been administered biotinylated bevacizumab or biotinylated denosumab following suture injury were subjected to streptavidin pull-down and immunoblotting for mouse FcγRI. Biotinylated bevacizumab, but not denosumab, interacted with mouse FcγRI in vivo. Anti-streptavidin immunoblotting confirmed efficient pull-down of both biotinylated antibodies. (b) FcγR humanized mouse corneas that had been administered bevacizumab or PBS following suture injury were subjected to immunoprecipitation of human FcγRI followed by immunoblotting for human IgG1 or phosphotyrosine. Bevacizumab, but not PBS, interacted with and induced phosphorylation of human FcγRI in vivo. Reprobing confirmed efficient immunoprecipitation of human FcγRI in both bevacizumab- and PBS-treated corneas. Each image is representative of three experiments (a, b). (c) Bevacizumab, but not PBS, increased Fcgr1 mRNA abundance in RAW264.7 mouse macrophages and in wild-type mouse corneas following suture injury, as monitored by real-time reverse transcription PCR, and FcγRI protein abundance in RAW264.7 cells, as monitored by western blotting. Densitometry of FcγRI normalized to Vinculin shown. n=4–6. Results are means±s.e.m. *P<0.05 compared with PBS.