Figure 2. Verification of tandem affinity purification (TAP) construct functionality. A–L: Subcellular localization of TAP protein fusions used in this study. S2 cells were transfected with pMK33-based constructs, induced overnight with 0.35 mM CuSO4, fixed, permeabilized with Triton X-100, and incubated with mouse IgG (Sigma), followed by incubation with Cy3-conjugated goat anti-mouse antibodies (Jackson). A,C,E,G,I,K: Nuclear staining (TO-PRO-3, Molecular Probes). B,D,F,H,J,L: Merged images. Amino-terminally TAP-tagged (NTAP)/Notch intracellular domain (NICD) was localized in the nucleus (E,F), similar to the control untagged NICD (A,B; Fehon et al., 1990). NTAP-Mam was also exclusively nuclear (I,J; Bettler et al., 1996). C,D,G,H,K,L: In contrast, full-length Notch-carboxy terminal TAP tag (NFL-CTAP) showed membrane and cytoplasmic staining (C,D; Fehon et al., 1990), whereas both NTAP-Deltex (NTAP-Dx; G,H) and Dx-carboxy terminal TAP tag (Dx-CTAP; K,L) were cytoplasmic (Busseau et al., 1994). M-R: Rescue of mutant phenotypes with TAP-tagged constructs. All crosses were set up at 25°C. M:nd3/Y; UAS-NFL-CTAP/CyO males showed characteristic notching at the wing tip. nd3 is a hypomorphic allele of N (Hing et al., 1994). N: In nd3/Y; UAS-NFL-CTAP/T113-GAL4 males, the wing notching was undetectable in 56–80% of all wings scored, depending on the particular transgenic line. This degree of rescue was similar to the rescue with an untagged full-length Notch (data not shown). O: A wing from an ec dx/Y; arm-GAL4/+ male, showing wing notching and widening of wing veins associated with the dx loss-of-function phenotype. P: This phenotype was rescued completely by expressing Dx-CTAP with the same driver (genotype ec dx/Y; arm-GAL4 UAS-Dx-CTAP/+). Q: Overexpression of a dominant-negative Mam isoform using the C96-GAL4 driver (Helms et al., 1999) resulted in severe wing notching around the wing margin (genotype C96-GAL4 UAS-MamH/+). R: Introduction of an NTAP-Mam construct suppressed this phenotype (UAS-NTAP-Mam/+; C96-GAL4 UAS-MamH/+).
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