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. 2010 Sep 7;123(19):3401–3411. doi: 10.1242/jcs.068684

Fig. 7.

Fig. 7.

Location of ESAG9-K69 protein in BSF K9:K69 cells engineered to express both ESAG9-K9 and ESAG9-K69 concurrently. (A) The ESAG9-K69 was detected using BB2 antibody and is visible in some cells as a signal from, or nearby, the flagellar pocket (white arrowheads) and also as a speckled staining on the slide itself. (B) Immunoprecipitation from cell lysate and conditioned media of monomorphic cells ectopically expressing ESAG9-K69. Immunoprecipitation was carried out with the BB2 antibody, which recognises the Ty epitope tag in the ESAG9-K69 sequence, in the presence (block) or absence (no block) of blocking peptide. Samples were analysed by western blotting using the BB2 antibody. There was a strong signal for ESAG9-K69 from the cell lysate in the absence of blocking peptide. (C) Immunoprecipitation from cell lysate (LY) and conditioned media (CM) of monomorphic cells ectopically expressing both ESAG9-K9 and ESAG9-K69. Immunoprecipitation was carried out with either BB2 antibody that recognises the Ty epitope tag in the ESAG9-K69 sequence, or anti-TbZFP3 antibody, which is an intracellular control, in the presence or absence of blocking peptides. Samples were analysed by western blotting using the respective antibodies. There was a strong signal for the intracellular control in the cell lysate in the absence of ZFP3 blocking peptide (no block), and no signal from the conditioned media. There was a strong signal for ESAG9-K69 from the conditioned media in the absence of blocking peptide and no signal from the cell lysate. Conditioned media from T. brucei EATRO 2340 stumpy form cells. (D) Stumpy form cells were harvested from a mouse 6 days post-innoculation and incubated in serum-free media. The conditioned media was then concentrated and analysed by western blotting with ESAG9-K69, Hsp70 (a control for cell integrity) and ESAG9-K9 antibodies. For all antibodies, there was a signal from both the cell lysate and concentrated conditioned media lanes. However, the signal from the CM lanes was much stronger for ESAG9-K69 and ESAG9-K9 than for Hsp70. Size markers are in kDa.