Figure 2

Establishing physiological levels of fusion protein expression. (a) A P-element insertion in the bithorax locus abolishes abdA expression and reproduces the expression profile of abdA. Compared to heterozygous embryos (upper panel), AbdA (abdominalA) expression (grey) is absent in embryos homozygous for the P insertion (named HC7JA1 [27]) that contains the β-galactosidase (β-Gal, red) reporter protein (bottom panel). (b) Establishing physiological levels of VCA expression with the armadillo (arm)-Gal4 driver. The average level of VCA expression at 29°C is quantified in the T2 thoracic segment and compared to the level of endogenous AbdA in the A2 segment of a wild type embryo (red-dotted circles). Fluorescent immunostainings were similarly performed with an anti-AbdA antibody (grey). Graph on the right is a boxplot representation of the statistical quantification of the surface and intensity of the fluorescent AbdA immunostaining (see also Methods). (c) Establishing physiological levels of expression with the abdA-Gal4 driver. Quantifications were measured with an anti-green fluorescent protein that recognises the VC fragment of VCA. Fluorescent immunostainings (grey) were performed in embryos expressing VCA either with arm-Gal4 or PabdA-Gal4 at 29°C. (d) The VC-AbdA (VCA) and VN-AbdA (VNA) fusion proteins are expressed at similar levels. Stage 10 embryos homozygous for the PabdA-Gal4 driver (symbolized by the exponent) and carrying one copy of VCA or VNA are stained with anti-AbdA antibody (grey). In these embryos, endogenous AbdA is absent, revealing the expression level of AbdA fusion proteins only. (e) The VN-extradenticle (Exd; VNE), VC-Exd (VCE) and Exd-VC (EVC) fusion proteins are expressed at similar levels. Exd fusion proteins are HA-tagged and were expressed with the abdA-Gal4 driver, as indicated. Graph on the right shows the level of fluorescent immunostaining of EVC (4) and VCE (5) when compared to levels of VNE. Fluorescent immunostaining (gray) against the HA tag was quantified as previously.