Western blot analysis of proNGF expression in DU145 and PC3 prostate cancer cell lysates. Monoclonal proNGF antibody M-1738-100 detects monomeric, non-glycosylated proNGF protein (20 ng, E.coli derived) at 25 kDa (Sample 1). PC3 (Sample 2, 100 µg) and DU145 (Sample 3, 100 µg) cell lysates show several proNGF isoforms (32 kDa, ~40 kDa, ~60 kDa, and higher molecular weight bands) as previously described, which most likely relates to glycosylated forms of full-length proNGF (Reinshagen et al., 2000; Lobos et al., 2005; Pedraza et al., 2005; Pundavela et al., 2014). Specificity of these bands is shown by pre-incubation with the immunizing peptide (5x mass of primary antibody), and a secondary antibody only control blot. Western Blotting Method: SDS-PAGE: denaturing and reducing, 4-20% Bis-Tris gel; Semi-Dry Transfer: Tris-Glycine (Towbin's) buffer with 20% methanol; Membrane: Nitrocellulose (0.45 µm); Blocking: 5% skim milk in TBST, 1 hour at RT; Primary antibody: 2 µg/mL, overnight at 4°C; Secondary antibody: anti-mouse-HRP (1/6000), 1 hour at RT; Detection: Chemiluminiscence.