CFB2 was expressed as a fusion with six His tags and NusA at the N-terminus, in BL12 Escherichia coli. Purified inclusion bodies were solubilized in 8 M urea and subjected to denaturing SDS-PAGE. The band containing 6×-His-NusA-CFB2 was used to raise antibodies in a rabbit. For affinity purification, membrane-bound 6×His-GB-CFB2 was used. (A) Coomassie-stained gel showing bacterial lysates, with or without induction of expression of either 6×His-GB-CFB2 or 6×His-Trx-CPSF73 (negative control). Serial dilutions of E. coli extracts are shown. A key for the different arrows is to the right at the top. The 100-fold dilution of 6×His-GB-CFB2 expressing cells (lane 3) gives roughly the same CFB2 band intensity as the markers (probably 1–5 µg each, data not available). The 100-fold dilution exceeds the Coomassie blue detection limit of ~100 ng. (B) CFB2 has relatively low abundance in bloodstream-form trypanosomes. The upper two panels are different exposures of a Western blot, and the third panel is the Ponceau red-stained membrane. In lane 1, lysate of 2 × 106 bloodstream forms expressing CFB2-6xmyc from an rRNA promoter was boiled in Laemmli buffer before loading; in lane 2, 107 bloodstream forms without tagged CFB2 expression were used and the extract was not boiled. Lane 3 is a 10−3× dilution of E. coli containing the 6×His-GB-CFB2 plasmid, but not induced; lane 4 is the same, but induced. Lanes 5–7 are successive 10-fold dilutions of the same extract. Lanes 8–11 are equivalent E.coli extracts, but the cells express 6×His-Trx-CPSF73; larger amounts of input protein were used because the recombinant protein was poorly expressed (see panel A). Lane 12 is an extract of Saccharomyces cerevisiae expressing AD-CFB2-myc; insufficient is present for detection. The top panel shows that the antibody interacts with 6×His-GB-CFB2, and not with 6×His-Trx-CPSF73, although there is a faint cross-reaction with a constitutively expressed E. coli protein of around 60 kDa. The purified antibody, which was raised against CFB2-NusA, is therefore interacting predominantly with CFB2 (trypanosomes do not contain NusA). The CFB2 band in lane 2 has approximately the same intensity as the 105-fold dilution of 6×His-GB-CFB2 E. coli lysate. From panel A, this dilution probably contains 1–10 ng of CFB2, which corresponds to between 1000 and 10,000 protein molecules per cell. (C) Purified anti-CFB2 antibody recognizes a band of the expected size that decreases after CFB2 RNAi and increases after proteasome inhibition. In lanes 1–6, 1 × 107 cells were lysed in sample buffer and loaded onto the gel. In lane 6 and also lane 7, which contains a lysate of E. coli expressing recombinant protein (CFB2-GB), the samples were boiled before loading. Lanes 1 and 6 are wild-type trypanosomes that had been incubated with MG132 for 1 hr. For lanes 2–5, cells with inducible RNAi were used, incubated with tetracycline inducer for the times indicated. The upper image was obtained after the membrane had been incubated with affinity-purified antibody (1/1000 dilution) for 1 hr. For the central panel, the membrane was cut to remove the region above 56 kDa that contains a cross-reacting band, then incubated overnight with the antibody. The lowest panel is the blotted membrane stained with Ponceau red. The key to the different arrows is above the membrane panels. (D) Cells used expressed various fusion proteins as shown in the top diagram. Separate Western blots from the same gel were used, with 2 × 106 loaded for detection of the myc epitope and 2 × 107 for CFB2. The affinity-purified anti-CFB2 antibody was incubated with the membrane overnight. Arrows are as in panel A. The cells expressing CFB2-6myc express VSG2 and the remaining cells express VSG22. (E) The effect of MG132 does not depend on an intact CFB2 N-terminus. Cells constitutively expressing CFB2 with 6myc tags at the N- or C-terminus were used, with or without a 1 hr incubation with MG132. The Western blot was incubated with anti-myc antibodies. The slower-migrating cross-reacting band also serves as a loading control.