FIG. 2.

Band II and other editing proteins. Western blotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. (A) Fractions from control trypanosome mitochondrial extract, resolved by glycerol gradient centrifugation and detected using antibody to band II. (B to D) Antibody to band II (B), band III (C), or band IV (D) was used to detect editing complex purified to the seven major proteins (32) (lanes “purif. complex”), small-scale extracts of two clonal 29.13 cell lines (ligase amounts were similar to those loaded from purified complex) (lanes “culture A” and “culture B”), and the respective recombinant protein (lanes “recomb band”). The recombinant proteins are larger than the corresponding native ones by the His₆ tag plus the mitochondrial targeting sequence. The antibodies do not appreciably detect either purified mitochondrial or recombinant versions of the other editing proteins. (E and F) Titrated amounts of small-scale trypanosome extract and recombinant band II (E) or band III (F) were detected using antibodies to the respective proteins. The recombinant protein was quantified as described by Bradford; ∼2/3 was full length. The trypanosome extract was 5 μg of protein/10⁷ cells; trypanosomes contain ∼50 μg of protein/10⁷ cells (17).