Figure 2.

Purification of the Mid1 protein and the presentation of its signal peptide. A, purification steps of the Mid1 protein tagged C-terminally with the FLAG epitope (Mid1-FL). Mid1-FL was purified by differential centrifugation, followed by immunoprecipitation with an anti-FLAG antibody. Proteins in an SDS-polyacrylamide gel were visualized with Coomassie Brilliant Blue. S12, P100, and X-S100 represent the supernatant of 12,000 × g centrifugation, the pellet of 100,000 × g centrifugation, and the supernatant of 100,000 × g centrifugation of a solubilized P100 preparation with Triton X-100. IP, immunoprecipitation. The arrow represents the position of Mid1-FL. B, Western blotting of immunoprecipitated samples with an anti-FLAG antibody. C, a signal peptide of Mid1 represented with an underline. The amino acid sequence LFQDF shown in red is that elucidated by amino acid sequencing as the N terminus of the purified, mature Mid1 protein. The arrowhead indicates the cleavage site between Gly²⁰ and Leu²¹.