Figure 2.
Proteins encoded by pre-(α)pro-GDNF and pre-(β)pro-GDNF cDNA localize to Golgi and DCVs in NGF-differentiated PC-6.3 cells. A–D, Localization of proteins encoded by human pre-(α)pro-GDNF or pre-(β)pro-GDNF cDNA in NGF-differentiated PC-6.3 cells (A). Double immunofluorescence stainings with anti-GDNF antibody (red) and anti-GM130 antibody for cis-to medial-Golgi marker protein (green). The GDNF-specific staining patterns detected are Golgi staining and vesicle-like staining. PlanApo 40×/1.00 (oil) objective (Olympus) was used. Scale bar, 10 μm. B, Quantification of localizations of the proteins encoded by pre-(α)pro-GDNF (gray bars) or pre-(β)pro-GDNF (black bars) in Golgi and in vesicle-like structures (n = 3). *p = 0.0023. Error bars show SD. C, IEM analysis of cells transfected with pre-(α)pro-GDNF (a, b, e) or pre-(β)pro-GDNF (c, d, f) cDNAs. In neurites, DCVs (arrows) are gathered nearby the plasma membrane (b, d). The anti-GDNF antibody-specific staining in DCVs (arrowheads) is shown in higher magnification (e, f). Small insets (a, c) show overview of the whole cell, in which magnified extension is marked (asterisk). Scale bars: a, c, 5 μm; b, d, 0.5 μm; e, f, 0.25 μm. D, IEM analysis of cells transfected with pre-(α)pro-GDNF (a, b, e) or pre-(β)pro-GDNF (c, d, f) cDNAs. Black clustered dots (arrows) indicate the localization of GDNF in Golgi complex (c, d). The anti-GDNF antibody-specific staining in Golgi complex transport vesicles is shown in higher magnification. Protein overexpression causes tubularization of the Golgi complex. Small insets (a, c) show overview of the whole cell, in which Golgi area is highlighted (asterisk). Nucleus (N) and Golgi complex (G) are indicated. Scale bars: a, c, 5 μm; b, d, 0.5 μm; e, f, 0.25 μm.