Figure 1.
Expression levels of α-synuclein in human non-melanoma cell lines.
Expression of endogenous α-synuclein was examined in different non-melanoma cancer cell lines by Western blotting using two different mouse monoclonal anti-α-synuclein antibodies (4D6 and 7B12.2). A total cell lysate of HeLa cells expressing FLAG-tagged α-synuclein was loaded as a positive control. To demonstrate expression of melanoma markers, we performed Western blotting using monoclonal antibodies against MART-1 and HMB-45. To demonstrate equal loading amounts of total cell lysates, Western blotting using anti-actin antibody was also performed (bottom panel). The detected proteins are indicated on the right. Molecular size markers are shown in kilodaltons.
Figure 2.
Expression levels of α-synuclein in cell lines of human brain tumors and retinoblastoma.
Expression of endogenous α-synuclein was examined in cell lines of human brain tumors and retinoblastoma by Western blotting using mouse monoclonal anti-α-synuclein antibody 4D6. Total cell lysates of HeLa cells expressing FLAG-tagged α-synuclein and SK-MEL28 cells were loaded as positive controls. To demonstrate expression of melanoma markers, we performed Western blotting using monoclonal antibodies against MART-1 and HMB-45. To demonstrate equal loading amounts of total cell lysates, Western blotting using anti-actin antibody was also performed (bottom panel). The detected proteins are indicated on the right. Molecular size markers are shown in kilodaltons.
Figure 3.
Expression levels of α-synuclein in human melanoma cell lines.
Expression of endogenous α-synuclein was examined in melanoma cell lines by Western blotting using mouse monoclonal anti-α-synuclein antibody 4D6. Total cell lysates of HeLa cells expressing FLAG-tagged α-synuclein and SK-MEL28 cells were loaded as positive controls. To demonstrate expression of melanoma markers, we performed Western blotting using monoclonal antibodies against MART-1 and HMB-45. To demonstrate equal loading amounts of total cell lysates, Western blotting using anti-actin antibody was also performed (bottom panel). The detected proteins are indicated on the right. Molecular size markers are shown in kilodaltons.
Table 1.
Summary of α-synuclein immunoreactivity in malignant melanoma.
Figure 4.
Expression of α-synuclein in human malignant melanoma tissues.
Tissue sections of 132 melanoma samples were immunostained with mouse monoclonal anti-α-synuclein antibody 4D6 (see text for identification of samples). The sections were then incubated with an alkaline phosphatase-conjugated anti-mouse IgG. After washing, Liquid Permanent Red substrate was used to develop the reaction and detect α-synuclein-positive cells. The sections were then counterstained with hematoxylin and analyzed by microscopy. The localization of α-synuclein is shown by the red color of Liquid Permanent Red substrate. Nuclear counterstaining is shown by the blue color of hematoxylin. Melanin pigments are brown in melanoma cells. Scale bar indicates 50 µm.
Figure 5.
Relationship of α-synuclein with MART-1 in human malignant melanoma tissues.
To investigate the relationship between α-synuclein and MART-1, tissue sections of melanoma samples (A and B) were double-immunostained with both mouse monoclonal anti-α-synuclein (4D6) antibody and rabbit monoclonal anti-MART-1 (EP1422Y) antibody. The sections were then labeled with Cy2-conjugated anti-mouse IgG for α-synuclein and Cy3-conjugated anti-rabbit IgG for MART-1. Finally, the sections were analyzed by fluorescence microscopy. The localization of α-synuclein is shown by the green fluorescence of Cy2 (panel a). The localization of MART-1 is shown by the red fluorescence of Cy3 (panel b). Their colocalization was examined by the merging of both fluorescent images (panel c). Scale bar indicates 20 µm.
Table 2.
Comparison between α-synuclein and MART-1 immunoreactivities in malignant melanoma.
Figure 6.
Relationship of α-synuclein with melanin pigments in human malignant melanoma tissues.
To investigate the relationship of α-synuclein with melanin pigments, tissue sections of pigmented melanoma were immunostained with mouse monoclonal anti-α-synuclein antibody 4D6. The sections were analyzed by fluorescence microscopy to determine the localization of α-synuclein and by bright field microscopy to determine the localization of melanin pigments. The localization of α-synuclein is shown by the green fluorescence of Cy2 (panel c). The localization of melanin pigments is shown by the brown color in the bright field microscopy (panel a). The localization of melanin pigments is shown by the red color in the dark field (panel b) because the image of brown pigments (panel a) was inverted and the color was then converted to red (panel b). In panel d, the image of panel b is merged with panel c. Scale bar indicates 20 µm.
Table 3.
Summary of α-synuclein immunoreactivity in nevi.
Figure 7.
Expression of α-synuclein in human nevus tissues.
Using mouse monoclonal anti-α-synuclein antibody 4D6, we immunostained tissue sections of 19 nevus samples (6 compound, 11 intradermal, and 2 junctional nevus samples) (see text for identification of samples). The sections were then incubated with an alkaline phosphatase-conjugated anti-mouse IgG. After washing, Liquid Permanent Red substrate was used to develop the reaction and detect α-synuclein-positive cells. The sections were then counterstained with hematoxylin and analyzed by microscopy. Six immunostainings are selected and shown in this figure. Panels A–C, compound nevus; Panels D–F, intradermal nevus. The localization of α-synuclein is shown by the red color of Liquid Permanent Red substrate. Nuclear counterstaining is shown by the blue color of hematoxylin. Melanin pigments are brown. Scale bar indicates 50 µm.
Table 4.
Summary of α-synuclein immunoreactivity in non-melanocytic cutaneous carcinoma.
Figure 8.
Expression of α-synuclein in tissues of patients with non-melanocytic cutaneous carcinoma.
Using mouse monoclonal anti-α-synuclein antibody 4D6, we immunostained skin sections of squamous cell carcinoma (6 samples) and basal cell carcinoma (5 samples), as well as malignant melanoma (positive control) and normal skin (negative control). The sections were then incubated with an alkaline phosphatase-conjugated anti-mouse IgG. After washing, Liquid Permanent Red substrate was used to develop the reaction and detect α-synuclein-positive cells. The sections were then counterstained with hematoxylin and analyzed by microscopy. For each malignant disease or normal skin, two immunostainings were selected and shown in this figure. Panels A and B, squamous cell carcinoma; Panels C and D, basal cell carcinoma; Panels E and F, malignant melanoma; Panels G and H, normal skin. The localization of α-synuclein is shown by the red color of Liquid Permanent Red substrate. Nuclear counterstaining is shown by the blue color of hematoxylin. Melanin pigments are brown. Scale bar indicates 50 µm.