Figure 7.
Identification of secreted forms of BDNFMet. Differentiated PC12 cells were transfected with BDNFVal (Val), BDNFMet (Met), or both BDNFVal and BDNFMet (Val/Met). The BDNF constructs contained either a single C-terminal HA epitope tag (A) or dual N-terminal HA and FLAG C-terminal tags (B). After 48 hr, media was collected under depolarization conditions, as described in Materials and Methods, in the absence or presence of protease inhibitors. BDNF in the media was immunoprecipitated with polyclonal anti-HA antibodies and analyzed by immunoblotting with monoclonal anti-HA antibodies. Representative blots from three independent experiments are shown.