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. 2014 Aug;13(8):1064–1076. doi: 10.1128/EC.00019-14

FIG 2.

FIG 2

Trypanosomal adenylate cyclases are glycosylated surface proteins that dimerize. (A) Western blot analysis of the proteins in the soluble (S) and insoluble pellet (P) fraction from cells extracted with Triton X-100 at the indicated temperature (4° or 37°C). Protein samples from cells expressing the indicated HA-tagged ACP (ACP1, ACP2, ACP4, or ACP5) were probed with antibodies against HA, calflagin, and tubulin. (B) Cells expressing HA-tagged ACP4 or ACP5 were surface biotinylated, and protein extracts harvested as indicated in the schematic were subjected to Western blot analysis with antibodies against HA or BiP. (C) Whole-cell lysates were prepared from 2913 control cells or cells expressing HA-tagged ACP1, ACP2, ACP4, or ACP5. Samples were untreated (−) or treated (+) with PNGase F and then subjected to Western blot analysis with antibodies against HA or EIF4AI. (D) Whole-cell lysates prepared from 2913 control cells or cells expressing HA-tagged ACP1, ACP2, ACP4, or ACP5 were separated by Blue Native gel electrophoresis and then transferred to a PVDF membrane and probed with anti-HA antibodies. (E) Cells with two tagged alleles of ACP1 (ACP1-Myc/HA) or two tagged alleles of ACP2 (ACP2-Myc/HA) were used for coimmunoprecipitation to test for homodimerization. In test samples, one allele of the indicated AC gene contains an HA tag and the other allele has a Myc tag. Samples were immunoprecipitated with anti-HA antibody, and input (I), unbound (U), and bound (B) fractions were probed in Western blots with anti-HA or anti-Myc antibody. As a negative control, ACP1-Myc lacks any HA tag and is not precipitated by anti-HA antibody (see Fig. S2 in the supplemental material). The numbers below each lane indicate the relative number of cell equivalents analyzed. (F) Cells expressing ACP1-HA were mixed with cells expressing ACP1-Myc, and the cells in the mixture were lysed and immunoprecipitated using anti-HA antibody. Input, unbound, and bound fractions were probed in Western blots with anti-HA or anti-Myc antibody.