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. 1997 Apr 7;137(1):51–65. doi: 10.1083/jcb.137.1.51

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Fractionation of leupeptin-treated A20 cells by free flow electrophoresis. (A) A20 cells were pulse labeled for 20 min and chased for 2 h in the presence of leupeptin before fractionation by FFE. Membranes collected in each fraction were pelleted by centrifugation and lysed in Triton X-100, and I-A^b class II molecules were immunoprecipitated using mAb MKD6. The samples were then analyzed by SDS-PAGE without boiling. The positions of compact dimers (“C”), α, β chains, Ii-p10, and a p12 protein of unknown origin are indicated relative to the positions of markers for the major protein peak (plasma membrane), endosomes/lysosomes (β-hexosaminidase), and anodally shifted CIIVcontaining fractions. (Left) Anode; (right) cathode. (B) I-A^b– expressing A20 cells were pulse labeled for 20 min and chased for 4 h in the presence of leupeptin before fractionation by FFE. The positions of p70 (Ii-p10–αβ complexes), 60-kD peptide-loaded compact dimers, free α and β chains, and Ii-p10 are indicated. (C) Positions of marker enzymes for plasma membrane (alkaline phosphodiesterase) and endosomes/lysosomes (β-hexosaminidase) in the FFE profile shown in B. p70 codistributed largely with the lysosomal marker β-hexosaminidase.

Fractionation of leupeptin-treated A20 cells by free flow electrophoresis. (A) A20 cells were pulse labeled for 20 min and chased for 2 h in the presence of leupeptin before fractionation by FFE. Membranes collected in each fraction were pelleted by centrifugation and lysed in Triton X-100, and I-A^b class II molecules were immunoprecipitated using mAb MKD6. The samples were then analyzed by SDS-PAGE without boiling. The positions of compact dimers (“C”), α, β chains, Ii-p10, and a p12 protein of unknown origin are indicated relative to the positions of markers for the major protein peak (plasma membrane), endosomes/lysosomes (β-hexosaminidase), and anodally shifted CIIVcontaining fractions. (Left) Anode; (right) cathode. (B) I-A^b– expressing A20 cells were pulse labeled for 20 min and chased for 4 h in the presence of leupeptin before fractionation by FFE. The positions of p70 (Ii-p10–αβ complexes), 60-kD peptide-loaded compact dimers, free α and β chains, and Ii-p10 are indicated. (C) Positions of marker enzymes for plasma membrane (alkaline phosphodiesterase) and endosomes/lysosomes (β-hexosaminidase) in the FFE profile shown in B. p70 codistributed largely with the lysosomal marker β-hexosaminidase.