Fig 1. Axin is localized at cell membrane-proximal puncta independently of Wingless pathway activation.

(A-I) Confocal images of third instar larval wing imaginal discs stained with antibodies indicated at bottom right; genotypes at left margin. (A-C) Wing disc stained with β-gal (A, magenta), Axin (B, green), and Arm (C, blue) antibodies. Axin¹⁸ null mutant clones (marked by the absence of β-gal, -/- in A) demonstrate the specificity of the Axin antibody. Armadillo marks the adherens junctions, which are present at the boundary between the apical and basolateral membrane, and also accumulates in the cytoplasm in Axin mutant clones. At this apical level, Axin staining is diffuse in the cytoplasm of all cells. (D-I) Axin staining at basolateral levels in the wing disc. Axin¹⁸ null mutant clones are marked by the absence of β-gal (D) or by dashed line (G). At this level, Axin antibody reveals specific staining that partially overlaps the basolateral membrane marker Fas III (I). Higher magnification views of the boxed area in (H) reveals endogenous Axin is present in puncta at or near the plasma membrane (G’-I’). Images were taken at the periphery of the wing discs. (J-L) Wild-type pupal wing (~28 hrs after pupa formation) double labeled with α-Dlg (J) and α-Axin (K). Endogenous Axin is also present in puncta proximal to the cell membrane in pupal wing (L). (M) Subcellular fractionation of lysates from S2R+ cells. The total lysates, cytoplasmic, and membrane fractions were analyzed by SDS-PAGE. Immunoblotting with Axin antibody revealed that Axin is present in both the cytoplasmic and membrane fractions. The efficiency of the fractionation was assayed by the presence of Arrow and Tubulin, membrane and cytoplasmic markers, respectively. (N) Quantification of the distribution of endogenous Axin in S2R+ cells. Results were obtained from four independent experiments, with a representative blot shown in (M). Values indicate mean ± SD. (O) Subcellular fractionation of lysates from 0–2.5 hour wild-type embryos. (P) Quantification of the distribution of endogenous Axin in 0–2.5 hour wild-type embryos. Results were obtained from four independent experiments, with a representative blot shown in (O). Values indicate mean ± SD. Scale bar: 5μm.