Immunoblotting experiments showed that both constructs were expressed in T. brucei procyclic forms (Fig. 7A, B, upper panels) and incorporated [3H]Etn (Fig. 7A, B, lower panels).
Both constructs were expressed in Cos7 cells, incubated with equivalent amounts of GST-tagged importin α3 or β1Δ(1 462) immobilized on glutathione beads, and then incubated with 80 µM RanQ69L-GTP.
In cells transfected with both wild type and MΔ5 constructs with or without IBV infection, detection of similar amounts of M protein was observed (Fig. 5b, top two panels, lanes 1 and 2), suggesting that both constructs were expressed at similar efficiencies.
Both constructs were expressed, purified, and compared with one another.
3 hr after electroporation, the embryos were screened based on fluorescence to ensure that both constructs were expressed and that the correct region was targeted for each construct.
Both constructs were expressed in E.coli BL21 DE3) cell lines by induction (at A600 ∼ 0.8) with 0.4 mM isopropyl-β d-thiogalactopyranoside (IPTG) at 37°C for 3 4 hr.
As expected, both constructs were expressed at a similar extent during early embryogenesis, as indicated by roughly comparable green fluorescence of embryos observed at the early blastula stage.
Intracellular staining using mAb 745H7 to the cytoplasmic portion of L1 monitored by flow cytometry showed that both constructs were expressed at comparable levels.
Both constructs were expressed at early stages of development (tail bud) while by 36 hpf the SQAS-GFP was not localized to somite boundary – there was weak fluorescence throughout the embryo – in contrast to Tsp4b-GFP.
Despite the fact that both constructs were expressed from pUB4 vector and under control of the same IND1 endogenous promoter, the truncated protein was present at a severely decreased level compared with wild-type Ind1 expression (ind1Δ + IND1).
