4D6 immunoreactivity with unphosphorylated and phosphorylated forms of α-syn. (A) 4D6 immunoreactivity with kinase-untreated and treated α-syn. Recombinant human α-syn was purified from bacterial culture and incubated with or without protein kinase CK2. Unphosphorylated and phosphorylated forms of α-syn were then analyzed by western blotting using anti-α-syn antibody LB509 (top panel), anti-phospho-α-syn antibody EP1536Y (middle panel) and anti-α-syn antibody 4D6 (bottom panel). Molecular size markers are shown in kilodaltons. (B) Specific immunoreactivity of 4D6 with S129-unphosphorylated form of α-syn expressed in SH-SY5Y cells. α-Syn (without tag) was overexpressed in SH-SY5Y cells by plasmid transfection. The total cell lysate was resolved by two-dimensional gel electrophoresis, and western-transferred to a membrane. Using a stripping method, the membrane was then serially probed with mouse monoclonal anti-α-syn antibody LB509 (top panel), rabbit monoclonal anti-phospho-α-syn antibody EP1536Y (middle panel) and mouse monoclonal anti-α-syn antibody 4D6 (bottom panel) for western blot analysis. Molecular size markers are shown in kilodaltons.

Subcellular localization of S129-unphosphorylated and phosphorylated forms of endogenous α-syn in human melanoma SK-MEL28 cells. (A) Double immunostaining of SK-MEL28 cells with anti-α-syn antibody 4D6 (for S129-unphosphorylated form) and anti-NUB1 antibody (for counterstaining). (B) Double immunostaining of SK-MEL28 cells with anti-phospho α-syn antibody pSyn#64 (for S129-phosphorylated form) and anti-NUB1 antibody (for counterstaining). (C) Comparison between 4D6 immunostaining and pSyn#64 immunostaining. Human melanoma SK-MEL28 cells were double-immunostained with mouse monoclonal anti-α-syn antibody (4D6 or pSyn#64) and rabbit polyclonal anti-NUB1 antibody. Cells were then labeled with fluorescent dye-conjugated secondary antibodies. The stained cells were analyzed by fluorescence microscopy. In A and B, the localization of endogenous α-syn is shown by the green fluorescence (left panels). The localization of endogenous NUB1 is shown by the red fluorescence. Both images are merged in the middle panels. The framed areas (middle panels) are magnified in the right panels. Scale bars indicate 20 µm in the left and middle panels and 5 µm in the right panels. In C, only merged images are shown with nuclear DAPI staining (the blue fluorescence). Scale bars indicate 20 µm.

Dot-like structures with phosphorylated α-syn around SK-MEL28 cells. Cells were immunostained with 4D6 for S129-unphosphorylated α-syn (A) or with pSyn#64 for S129-phosphorylated α-syn (B). Cells were then labeled with Alexa Fluor 488-conjugated secondary antibody. The stained cells were analyzed by fluorescence microscopy with longer exposure to demonstrate extracellular dot-like structures. The localization of endogenous α-syn is shown by the green fluorescence of Alexa Fluor 488. The framed areas (left panels) are magnified in the right panels. Scale bars indicate 20 µm in the left and 5 µm in the right panels.

Immunoreactivity of 4D6 and pSyn#64 antibodies to α-syn-knockdown SK-MEL28 cells. (A) Expression levels of α-syn in SK-MEL28 cells expressing shRNA of α-syn. Total cell lysates were prepared from cells expressing control shRNA (first lane) and cells expressing α-syn shRNA (no. 1–no. 5). These lysates were analyzed by western blotting using anti-α-syn antibody 4D6 (upper panel) and anti-actin antibody (lower panel). Molecular size markers are shown in kilodaltons. (B) Immunostaining of α-syn-knockdown SK-MEL28 cells. Cells expressing control shRNA (left panels) and those expressing α-syn shRNA (right panels) were immunostained with anti-α-syn antibodies 4D6 (upper panels) and pSyn#64 (lower panels). The localization of α-syn is shown by the green fluorescence. Nuclei are counterstained blue by DAPI. Scale bars indicate 50 µm.

Electron microscopy for ultrastructure of SK-MEL28 cells. Cells were postfixed in osmium tetroxide, stained en bloc with uranyl acetate, and embedded in epon-araldite resin. Ultrathin sections were cut and sequentially stained with uranyl acetate and lead citrate. The sections were observed by a transmission electron microscope. The framed areas in A are magnified in B and C. The framed area in D is magnified in (E). Scale bars indicate 2 µm in A and D and 400 nm in B, C, and E. Arrows in B and E indicate MVs on the cell surface. An arrowhead in C indicates the vesicular ‘budding’ or ‘fusion’ on the cell surface.

Ultrastructural localization of S129-phosphorylated, endogenous α-syn in SK-MEL28 cells. Immuno-colloidal gold electron microscopy was performed using an antibody to S129-phosphorylated α-syn (pSyn#64). Thin sections of SK-MEL28 cells were sequentially incubated with pSyn#64 and secondary antibody conjugated with colloidal gold particles (15 nm in diameter). After staining with uranyl acetate, the sections were observed by a transmission electron microscope. (A) Vertical section showing α-syn clusters or oligomers in the cytoplasm. (B–D) Sections showing MVs containing α-syn. Scale bars indicate 200 nm. Arrowheads indicate microvilli on the cell surface. Arrows indicate the localization of α-syn labeled with colloidal gold particles.

Localization of S129-unphosphorylated and phosphorylated forms of endogenous α-syn in various human melanoma cell lines. (A) Expression levels of S129-unphosphorylated and phosphorylated α-syn. Total cell lysates were prepared from human melanoma cell lines, SK-MEL28, SK-MEL5, A375, MeWo and WM266-4, and human lung fibrosarcoma cell line HT1080 (negative control). The lysates were analyzed by western blotting using anti-α-syn antibodies LB509 (for total α-syn), 4D6 (for S129-unphosphorylated form), pSyn#64 (for S129-phosphorylated form) and EP1536Y (for S129-phosphorylated form). In addition, expression levels of NUB1 and actin were examined. Molecular size markers are shown in kilodaltons. (B) Double immunostaining of HT1080 and three melanoma cell lines with anti-α-syn antibody (4D6 or pSyn#64) and anti-NUB1 antibody (for counterstaining). The localization of endogenous α-syn is shown by the green fluorescence. The localization of endogenous NUB1 is shown by the red fluorescence. Nuclear counterstaining is shown by the blue fluorescence of DAPI. These three-color images are merged in all panels. The framed areas (middle panels) are magnified in the bottom panels. Scale bars indicate 20 µm in the top and middle panels and 5 µm in the bottom panels.