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. 1998 Nov 16;143(4):947–955. doi: 10.1083/jcb.143.4.947

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(A) Immunoadsorption of SVs derived from the plasma membrane. Aliquots of SVs were incubated with M-450 Dynabeads (3 mg per reaction) coated with anti-VAMP (synaptobrevin), anti-synaptophysin (SY-38), anti-synaptogyrin (p29), anti-SV2, or anti-syntaxin antibodies. Mouse γ-globulin was used to determine non-specific binding. After extensive wash, the radioactivity associated with the beads was plotted as a percentage of total radioactivity for each reaction. (B and C) TfRs are sorted from SVs from plasma membrane. N49A/PC12 cells were labeled with ¹²⁵I-Tf (0.2 μg/ml; B) or ¹²⁵I-KT3 (10 μg/ml; C) at 4°C for 2 h. The cell homogenate was incubated with rat brain cytosol and ATP at 37°C (closed symbols) or 4°C (open symbols). A low speed (5,000 g min) supernatant was made from each reaction and centrifuged on a 10– 45% sucrose velocity gradient. Radioactivity of each gradient fraction was plotted against its sucrose concentration. Radioactivity from ¹²⁵I-KT3 was recovered at 23% (1.5 sucrose; closed diamonds), similar to the SVs generated from endosomes (Lichtenstein et al., 1998). However, ¹²⁵I-Tf–labeled membranes were recovered at 28% (0.5 sucrose; closed circles), well separated from the SVs.

(A) Immunoadsorption of SVs derived from the plasma membrane. Aliquots of SVs were incubated with M-450 Dynabeads (3 mg per reaction) coated with anti-VAMP (synaptobrevin), anti-synaptophysin (SY-38), anti-synaptogyrin (p29), anti-SV2, or anti-syntaxin antibodies. Mouse γ-globulin was used to determine non-specific binding. After extensive wash, the radioactivity associated with the beads was plotted as a percentage of total radioactivity for each reaction. (B and C) TfRs are sorted from SVs from plasma membrane. N49A/PC12 cells were labeled with ¹²⁵I-Tf (0.2 μg/ml; B) or ¹²⁵I-KT3 (10 μg/ml; C) at 4°C for 2 h. The cell homogenate was incubated with rat brain cytosol and ATP at 37°C (closed symbols) or 4°C (open symbols). A low speed (5,000 g min) supernatant was made from each reaction and centrifuged on a 10– 45% sucrose velocity gradient. Radioactivity of each gradient fraction was plotted against its sucrose concentration. Radioactivity from ¹²⁵I-KT3 was recovered at 23% (1.5 sucrose; closed diamonds), similar to the SVs generated from endosomes (Lichtenstein et al., 1998). However, ¹²⁵I-Tf–labeled membranes were recovered at 28% (0.5 sucrose; closed circles), well separated from the SVs.