Figure 7.
Daple directly interacted with apical MTs to show planar polarization. (A) Co-IP of Daple with α-tubulin (αtub). mEGFP–Daple FL was exogenously overexpressed in HEK293T cells and pulled down using an anti–α-tubulin antibody. IB, immunoblotting. (B) MT co-sedimentation assay with purified mEGFP–Daple FL protein. Coomassie Blue–stained SDS-PAGE gels showing the binding behavior of 500 nM mEGFP–Daple FL in the presence of increasing concentrations of MTs (upper panel). Results from three experiments were plotted and fit to a Michaelis-Menten equation (Kd ± SD, 1.46 ± 0.187 µM for Daple FL; lower panel). S, supernatant; P, pellet. (C) MT co-sedimentation assay with purified mEGFP–Daple CT and NT domains and mEGFP–Daple FL ΔCT. Schematic illustration of Daple CT and NT domains and Daple ΔCT (upper panel). Coomassie Blue–stained SDS-PAGE gels (lower panel) showing the binding behavior of mEGFP–Daple CT (750 nM), mEGFP–Daple NT (1.2 µM), and mEGFP–Daple ΔCT (480 nM) with 10 µM MTs. Red arrows show Daple and its indicated domains. (D) Schematic illustration of the Daple CT domain (upper panel) and representative images of WT MTECs expressing mEGFP–Daple CT and the CT domain lacking Fzd-binding domain (1868–2006 aa; CT ΔFzd-binding domain) via a lentivirus system (lower panel). mEGFP (green), Fzd6 (magenta), and α-tubulin (blue). Arrows indicate the asymmetrical localization of Fzd6 and CT at the AJC. (E) Co-IP of the mEGFP–Daple 1390–1867 or 1868–2009 domains with α-tubulin. mEGFP-Daple domains were exogenously overexpressed in HEK293T cells and pulled down with an anti–α-tubulin antibody. Red arrows show Daple and its indicated domains. (F) Representative immunofluorescence images of mEGFP–Daple FL Δ1390–1867 (magenta) and α-tubulin (green) in Daple-KO MTECs expressing mEGFP–Daple FL Δ1390–1867 via a lentivirus system. The intensity of the α-tubulin signal is represented by the color map (red indicates high intensity; blue indicates low intensity). (G) Statistical analyses of the slope coefficients (Coef.) acquired from the intensity profiles of the immunofluorescence for α-tubulin along the linear ROI set either from the opposite to the Fzd6 side of the AJC in mEGFP-positive MCCs (orange) or arbitrarily between one side and the opposite side of the AJC in mEGFP-negative MCCs (blue) of Daple-KO MTECs expressing mEGFP–Daple FL Δ1390–1867 via a lentivirus system (n = 6 cells). Two-tailed Mann-Whitney U test; n.s., P ≥ 0.05. (H) Representative immunofluorescence images of Daple-KO MTECs expressing the mEGFP–Daple CT domain via a lentivirus system for signals of mEGFP (magenta) and α-tubulin (green). The intensity of the α-tubulin signal is represented by the color map (red indicates high intensity; blue indicates low intensity). (I) Statistical analyses of the slope coefficients acquired from the intensity profiles of the immunofluorescence intensities for α-tubulin along the linear ROI set either from the opposite side of the Fzd side of the AJC to the Fzd6 side of the AJC in mEGFP-positive MCCs (orange) or arbitrarily between one side and the opposite side of the AJC in mEGFP-negative MCCs (blue) of Daple-KO MTECs expressing the mEGFP–Daple CT domain via a lentivirus system (n = 6 cells). Two-tailed Mann-Whitney U test; n.s., P ≥ 0.05. Scale bars represent 5 µm.

Daple directly interacted with apical MTs to show planar polarization. (A) Co-IP of Daple with α-tubulin (αtub). mEGFP–Daple FL was exogenously overexpressed in HEK293T cells and pulled down using an anti–α-tubulin antibody. IB, immunoblotting. (B) MT co-sedimentation assay with purified mEGFP–Daple FL protein. Coomassie Blue–stained SDS-PAGE gels showing the binding behavior of 500 nM mEGFP–Daple FL in the presence of increasing concentrations of MTs (upper panel). Results from three experiments were plotted and fit to a Michaelis-Menten equation (Kd ± SD, 1.46 ± 0.187 µM for Daple FL; lower panel). S, supernatant; P, pellet. (C) MT co-sedimentation assay with purified mEGFP–Daple CT and NT domains and mEGFP–Daple FL ΔCT. Schematic illustration of Daple CT and NT domains and Daple ΔCT (upper panel). Coomassie Blue–stained SDS-PAGE gels (lower panel) showing the binding behavior of mEGFP–Daple CT (750 nM), mEGFP–Daple NT (1.2 µM), and mEGFP–Daple ΔCT (480 nM) with 10 µM MTs. Red arrows show Daple and its indicated domains. (D) Schematic illustration of the Daple CT domain (upper panel) and representative images of WT MTECs expressing mEGFP–Daple CT and the CT domain lacking Fzd-binding domain (1868–2006 aa; CT ΔFzd-binding domain) via a lentivirus system (lower panel). mEGFP (green), Fzd6 (magenta), and α-tubulin (blue). Arrows indicate the asymmetrical localization of Fzd6 and CT at the AJC. (E) Co-IP of the mEGFP–Daple 1390–1867 or 1868–2009 domains with α-tubulin. mEGFP-Daple domains were exogenously overexpressed in HEK293T cells and pulled down with an anti–α-tubulin antibody. Red arrows show Daple and its indicated domains. (F) Representative immunofluorescence images of mEGFP–Daple FL Δ1390–1867 (magenta) and α-tubulin (green) in Daple-KO MTECs expressing mEGFP–Daple FL Δ1390–1867 via a lentivirus system. The intensity of the α-tubulin signal is represented by the color map (red indicates high intensity; blue indicates low intensity). (G) Statistical analyses of the slope coefficients (Coef.) acquired from the intensity profiles of the immunofluorescence for α-tubulin along the linear ROI set either from the opposite to the Fzd6 side of the AJC in mEGFP-positive MCCs (orange) or arbitrarily between one side and the opposite side of the AJC in mEGFP-negative MCCs (blue) of Daple-KO MTECs expressing mEGFP–Daple FL Δ1390–1867 via a lentivirus system (n = 6 cells). Two-tailed Mann-Whitney U test; n.s., P ≥ 0.05. (H) Representative immunofluorescence images of Daple-KO MTECs expressing the mEGFP–Daple CT domain via a lentivirus system for signals of mEGFP (magenta) and α-tubulin (green). The intensity of the α-tubulin signal is represented by the color map (red indicates high intensity; blue indicates low intensity). (I) Statistical analyses of the slope coefficients acquired from the intensity profiles of the immunofluorescence intensities for α-tubulin along the linear ROI set either from the opposite side of the Fzd side of the AJC to the Fzd6 side of the AJC in mEGFP-positive MCCs (orange) or arbitrarily between one side and the opposite side of the AJC in mEGFP-negative MCCs (blue) of Daple-KO MTECs expressing the mEGFP–Daple CT domain via a lentivirus system (n = 6 cells). Two-tailed Mann-Whitney U test; n.s., P ≥ 0.05. Scale bars represent 5 µm.

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