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FIG. 4.

FIG. 4. From: A Functional Domain of Dof That Is Required for Fibroblast Growth Factor Signaling .

Subcellular localization of mutant Dof proteins and influence of Dof on localization of ectopically expressed Htl. (A to L) The stabilities and subcellular localization of mutant forms of Dof were assessed using a prd-Gal4 transgene to express each construct in ectodermal cells. The expression and subcellular localization of the mutant proteins were followed by staining fixed embryos with an anti-Dof antiserum. (M to O) A transgene encoding Htl was expressed using prd-Gal4 either alone (M) or in combination with a transgene encoding Dof with an N-terminal flag epitope tag (N and O). The subcellular distribution of Htl (M and N) was visualized by staining the embryos with an anti-Htl antiserum, and the localization of Dof (O) was determined as described above. All embryos depicted are of the same age (stage 10). The orientations of the embryos are shown in the top right corner of each panel; the white boxes indicate the regions (39 by 35 μm) that were photographed at high magnification. The proteins ectopically expressed are indicated in each panel.

Robert Wilson, et al. Mol Cell Biol. 2004 March;24(6):2263-2276.
FIG. 2.

FIG. 2. From: A Functional Domain of Dof That Is Required for Fibroblast Growth Factor Signaling .

Abilities of different mutant Dof proteins to rescue defects in the development of the mesoderm and tracheae. (A to K) Tubular network of the tracheae, revealed using the monoclonal antibody 2A12, is shown in brown. Even-skipped, visualized as a blue stain, was used to identify the homozygous mutant embryos by the absence of pericardial cells expressing Eve in the mesoderm, which can be seen in the wild-type embryo in panel A as a dorsal row of faint out-of-focus spots. (A) Stage 15 wild-type embryo; (B) homozygous dof1 mutant embryo. (C to K). Tracheal development of homozygous dof1 mutant embryos in which mutant forms of Dof were overexpressed using a btl-Gal4 transgene. The embryos chosen for flag-Dof[1-674] (E), Dof[1-446] (F), Dof[89-1012] (G), flag-Dof[Δ361-449] (I), and SH4-Dof (K) show the typical rescues obtained with these constructs, while those chosen for flag-Dof[Δ233-449] (H) and flag-Dof[Δ233-364] (J) show the best rescue of tracheal development obtained with these constructs. (L to O) Abilities of the mutant Dof proteins flag-Dof (L), Dof[1-522] (M), Dof[89-1012] (N), and Dof[1-446] (O) to promote differentiation of the mesoderm in stage 11 embryos were determined by monitoring the expression of Even-skipped, shown in brown, in parasegments 3 to 13. The embryos shown are representative, although a large degree of variation in the number of Even-skipped positive clusters was observed for the mutant Dof[89-1012].

Robert Wilson, et al. Mol Cell Biol. 2004 March;24(6):2263-2276.

FIG.1. From: A Functional Domain of Dof That Is Required for Fibroblast Growth Factor Signaling .

Sequence analysis of Dof and related proteins. (A) Aligment over the DBB motif and the ankyrin repeats of Drosophila Dof (accession number O96757), Anopheles Dof (accession number Q8T5J9), mouse BCAP (accession number Q9EQ32), chicken BCAP (accession number AAG48583), human BCAP (accession number XP_058343), fugu BCAP (ENSEMBL SINFRUP00000078060), lamprey BCAP (accession number AAN64296), human BANK (accession number Q8WYN5), and Ciona intestinalis BCAP (a translation of accession number AK115423). The alignment begins at positions 216 of Drosophila Dof, 395 of Anopheles Dof, 182 of mouse BCAP, 3 of human BCAP, 180 of the predicted fugu BCAP sequence, 178 of lamprey BCAP, 170 of human BANK, and 1 of the incomplete Ciona BCAP sequence. Six or more identical or similar residues shared by the sequences are shown in red and blue, respectively, and are marked below the alignment by a dot. An asterisk denotes a position that is absolutely conserved. (B) Alignment of A. gambiae Dof and Drosophila Dof was made using CLUSTAL W, and regions representing insertions in the Anopheles sequence were removed. Then, the number of identities found within a window of 15 amino acids was plotted for each position of the Drosophila protein. The locations of the DBB motif, the ankyrin repeats, and the coiled coil within Drosophila Dof are indicated at the top of the chart. The positions of insertions and deletions within the Anopheles sequence are shown below the chart as arrows pointing toward and away from the sequence of the Drosophila protein, respectively. The numbers represent the lengths of the insertions and deletions.

Robert Wilson, et al. Mol Cell Biol. 2004 March;24(6):2263-2276.
FIG. 3.

FIG. 3. From: A Functional Domain of Dof That Is Required for Fibroblast Growth Factor Signaling .

Functions of mutant Dof proteins in in vivo assays. The Dof constructs are shown schematically on the left. The amino acids present in each construct are noted. A flag epitope tag is represented by a red circle, while the membrane-targeting SH4 domain of human Src is shown as a blue triangle. The extracellular portion and transmembrane domain of the Torso mutant 4021 is shown as a green diamond (also see Fig. 6 for details). To assess the ability of a transgene to rescue tracheal development, we counted the junctions formed by the dorsal trunk in five stage 15 embryos. In the case of the mesoderm, we determined the number of Eve-positive clusters formed in five stage 11 embryos. The degree of variability of the data is depicted as a box-and-whiskers plot. A thick vertical line indicates the median value, a horizontal shaded box represents the interquartile range between the first and third quartiles, and a thin horizontal line represents the entire range of the data. For the mutants flag-Dof[1-674], Dof[1-522], Dof[168-1012], flag-Dof[Δ361-449], SH4-Dof, flag-Dof <Y486F>, and flag-Dof[1-802]<Y97F, Y486F, Y515F>, two independent transgenic lines were assessed in the tracheae. In most cases, the absence of a dorsal trunk reflected a complete failure of the transgene to rescue the homozygous dof1 mutant phenotype (cf. Fig. 2). The asterisks shown for the mutants flag-Dof[Δ233-449] and flag-Dof[Δ233-364] indicate the formation of some lateral trunk (Fig. 2H and J).

Robert Wilson, et al. Mol Cell Biol. 2004 March;24(6):2263-2276.
FIG. 6.

FIG. 6. From: A Functional Domain of Dof That Is Required for Fibroblast Growth Factor Signaling .

Dof phosphorylation in response to FGF receptor activation and the effect of specific tyrosine mutations on the function of Dof. (A) Drosophila Schneider S2 cell lines were stably transformed as indicated above each lane with different combinations of an activated form of the FGF receptor Btl under the control of a heat shock promoter (λ-Btl) and Dof with an N-terminal FLAG epitope tag under the control of the actin5C promoter (FLAG-Dof). The presence of Dof and proteins with phosphorylated tyrosine residues in whole-cell lysates of these cell lines, previously induced to express the FGF receptor (left), or immunoprecipitates (IP) of the induced lysates with an antiserum directed against Dof (α-Dof) (right) was monitored by Western blotting with antibodies directed against the FLAG epitope tag (M5) (α-FLAG) and phosphotyrosine (4G10) (α-PY). The sizes of the proteins detected are indicated on the left. Dof is seen at an apparent molecular mass of ∼130 kDa. Often, an additional band representing a breakdown product is seen at ∼70 kDa. (B) Schematic representations of Dof constructs. The name of a signaling protein and a Y indicates the position of a tyrosine in Dof that is a potential binding site for this molecule. An F indicates a specific mutation that results in a protein containing a phenylalanine residue rather than a tyrosine; different combinations of mutations in amino acids 97, 486, and 515 are shown. (C to H) Tracheal development of homozygous dof mutant embryos expressing the constructs flag-Dof <Y95F> (C), flag-Dof <Y486F> (D), flag-Dof <Y515F> (E), flag-Dof <Y486F, Y515F> (F), flag-Dof[1-802] <Y97F> (G), and flag-Dof[1-802] <Y97F, Y486F, Y515F> (H). The embryos are stained for 2A12 in brown and Even-skipped in blue to distinguish the tracheae and the genotype, respectively, as described in the legend to Fig. 2.

Robert Wilson, et al. Mol Cell Biol. 2004 March;24(6):2263-2276.
FIG. 5.

FIG. 5. From: A Functional Domain of Dof That Is Required for Fibroblast Growth Factor Signaling .

Interaction of the FGF receptor Heartless with mutant forms of Dof. (A) Diagram of Heartless (accession number Q07407) showing the transmembrane domain and the ATP binding and autophosphorylation sites. The two blue bars represent the longest and shortest regions encoded by heartless clones isolated in a yeast two-hybrid screen with Dof (the fusion of the Gal4 activation domain with Htl corresponds to amino acids 349 and 491, respectively). TM, transmembrane region. (B) Coimmunoprecipitation of Dof with the FGF receptor Htl. Lysates from Schneider S2 cells coexpressing a constitutively active, hemagglutinin (HA)-tagged form of Htl (lambda-Htl) and a Flag-tagged full-length Dof protein were used for immunoprecipitation (IP) with antibody against FLAG, Dof, or HA or control beads without antibodies. The immunoprecipitates were analyzed by SDS-PAGE together with the whole-cell lysate (input). Western blots were stained with antibodies against Flag to visualize Dof (left) and against HA to visualize the FGF receptor (right). In addition to the band corresponding to the full-length Flag-tagged Dof molecule at ∼130 kDa, a breakdown product at 70 kDa is usually seen. (C and D) Growth of yeast clones coexpressing Dof mutants fused to the Gal4 DNA binding domain (DB) and fragments of Htl fused to the Gal4 activation domain (AD). The different Dof mutants tested are shown schematically on the left (see Fig. 3 for details). Serial dilutions of cell suspensions were spotted onto plates containing the indicated selection media (His selection, SC-H, or the more stringent Ade selection, SC-ULA) and grown for 3 days. In panel D, the results of the comparison of the behavior of the long fragment of Htl under the two selection conditions are shown schematically. +, strong growth at all three densities; −, no detectable growth at the lower densities and minimal or no growth at the highest density. (E) Western blots of whole-cell lysates (input) and immunoprecipitations from Schneider S2 cells expressing a constitutively active HA-tagged FGF receptor (λHtl) and Flag-tagged mutated versions of Dof. Protein complexes were immunoprecipitated from the lysates using antibody against Dof, Flag, or HA, and the precipitated proteins were detected on the Western blots using antibody against Flag or HA. The asterisk marks the immunoglobulin G heavy chain of the Flag antibody used for immunoprecipitation. In addition to a protein of approximately the size predicted for each of the constructs, in each lane there is also a smaller fragment representing a breakdown product. The sizes of these smaller fragments are in all cases consistent with a cleavage of Dof at the same position between the ankyrin repeat and the coiled-coil region. The five top panels are from the same experiment. The bottom panel is from a separate experiment to show the variation in the amount of DofΔ233-449 associated with the receptor. The expression levels in this experiment were the same as in experiment (Exp.) I. Note that although Dof[168-1012] is not detectable in the whole-cell lysate at this exposure, a certain amount is precipitable and (in experiment II) is able to coprecipitate the receptor.

Robert Wilson, et al. Mol Cell Biol. 2004 March;24(6):2263-2276.

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