Figure 1.

Characterization of anti-proNT-3 antiserum. A, Sf9 cells expressing full-length preproNT-3 cDNA synthesize both proNT-3 and mature NT-3 as indicated (red arrows). Total cellular lysates, along with recombinant NT-3 standard (Promega), were Western blotted with an anti-NT-3 antiserum (sc-547; Santa Cruz Biotechnology) followed by reprobing the blot with an anti-proNT-3 antiserum (see Materials and Methods) to demonstrate the specificity of the prodomain specific antiserum. The numbers on the left indicate positions of the molecular weight markers. B, Western blot of Sf9 cells expressing proNGF, proBDNF, or proNT-3 using preimmune or affinity-purified proNT-3 specific antibody as indicated. Note that the anti-proNT-3 antiserum does not recognize proNGF or proBDNF. Reprobing the blot with anti-NGF and anti-BDNF antisera confirmed the expression of the corresponding proneurotrophins in the appropriate cell lysates (data not shown). The red arrow on the right indicates position of proNT-3, and the numbers on the left indicate positions of the molecular weight markers. C, HEK 293T cells were cotransfected with plasmids encoding GFP (to identify transfected cells) and proNT-3, proBDNF, or proNGF as indicated. Forty-eight hours later, cells were fixed and indirect immunofluorescence microscopy was performed using preimmune serum or the affinity-purified anti-proNT-3 antibody. The top panels represent merged images (with DAPI) of GFP epifluorescence and preimmune or proNT-3 immunoreactivity (bottom insets) from the corresponding transfected cells.