Quantitative Immunoblot Analysis of Nerve Injury

Immunoblot analysis was performed as described previously (Campana et al., 2006a (link); Inoue et al., 2010 (link)). Briefly, extracts of sciatic nerve tissue distal to the crush injury site were prepared in RIPA buffer. The protein content of each extract was determined by bicinchoninic acid (BCA) assay. An equivalent amount of protein (20–40 μg per lane) was subjected to 10% SDS-PAGE and electro-transferred to nitrocellulose membranes. Blots were blocked with 5% nonfat dry milk and subsequently incubated with primary polyclonal antibodies to GAP43 (1:1000; Sigma), STMN2 (1:1000; Novus Biologicals), total ERK1/2 (t-ERK) (1:1000; Cell Signaling), GAPDH (1:2000; Sigma), NF200 (1:800; Sigma), p75^NTR (1:5000; Millipore) or βIII neuronal tubulin (Tuji; 1:10,000; BioLegend) overnight at 4° C. Antibody-binding was detected by HRP-conjugated species-specific secondary antibodies (1:2000; Cell Signaling) and enhanced chemiluminescence (GE Healthcare). Blots were scanned (Canoscan) and densitometry was performed using Image J software.