Fig. 9. TGN46 localizes to vacuoles and to multivesicular bodies in C312-overexpressing cells. HeLa cells transfected with expression vectors for Tac and for (A) C312(Y2187A) or (B-F) C312 were sorted for Tac cell surface staining. Positive cells were exposed to HRP for 15-30 minutes at 37°C, then fixed, and ultrathin cryosections were labeled with antibodies to HA, TGN46 and or HRP and gold-conjugated secondary antibodies for immuno-EM analyses. (A-C) Sections were labeled with anti-HA and 10 nm gold-conjugated anti-mouse immunoglobulin, and anti-TGN46 and 5 nm gold-conjugated anti-sheep immunoglobulin. Arrowheads in B and C point to 5 nm gold particles. Note the labeling of the trans face of the Golgi complex by both anti-HA and anti-TGN46 in A, and the dense labeling of vacuoles with anti-HA in B and C. These examples show TGN46 in the vacuolated structures. (D-F) Sections were labeled with anti-HRP and 10 nm gold-conjugated anti-rabbit immunoglobulin, and anti-TGN46 and 5 nm gold-conjugated anti-sheep immunoglobulin. Arrowheads point to 5 nm gold particles (TGN46) in multivesicular endosomes (mve) that are also labeled by anti-HRP (10 nm gold). Bars, 0.1 µm.

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