Fig. 7

APC2 clustering is driven by DYNLL2 binding. a Neurons were transfected with RFP-APC2 and either GFP, GFP-DYNLL2, GFP-DYNLL1 or GFP-DYNC1LI2 or RFP and GFP-DYNLL2 and imaged 24 h later. b Streptavidin pull-down assay with BioGFP-DYNLL2 and the indicated APC2-C truncations, expressed in HEK293T cell lysates, analyzed by Western blotting with the indicated antibodies. c Streptavidin pull-down assay with BioGFP-DYNLL1 and the indicated APC2-C truncations, expressed in HEK293T cell lysates, analyzed by Western blotting with the indicated antibodies. d Streptavidin pull-down assay with BioGFP-DYNC1H1 and the indicated APC2-C truncations, expressed in HEK293T cell lysates, analyzed by Western blotting with the indicated antibodies. e Cos7 cells were transfected with GFP-DYNLL2 and either RFP, RFP-APC2, RFP-APC2-C, RFP-ACP2-C(c1AAA) mutation or RFP-ACP2-C(c2AAA) mutation, fixed 24 h later and imaged. GFP-DYNLL2 co-transfected with RFP-APC2 and imaged live after 24 h. f Thresholded Manders’ coefficient was measured from max two areas per cell in 10 × 10 μm ROIs. (n = 25 areas for RFP-APC2, n = 32 areas for RFP-APC2-C, n = 30 areas for RFP-APC2-C(c1AAA), n = 35 areas for RFP-APC2-C(c2AAA), N = 2, One-Way ANOVA, Tukey post-hoc). g Streptavidin pull-down assay with BioGFP-DYNLL2 and the indicated C-terminus mutations, expressed in HEK293T cell lysates, analyzed by Western blotting with the indicated antibodies. h Neurons were transfected with either RFP-APC2 or RFP-APC2(c2AAA) and dendrites were imaged 24 h later at DIV15–16. i Sholl analysis of dendritic branching at DIV11 (n = 34 neurons control shRNA + RFP, n = 35 neurons APC2 shRNA + RFP, n = 28 neurons APC2 shRNA + RFP-APC2, n = 34 neurons APC2 shRNA + RFP-APC2-ΔC, n = 20 neurons APC2 shRNA + RFP-APC2(c2AAA), N = 3, Two-Way ANOVA, Tukey post-hoc). j Model for APC2 interaction with microtubules (schematic drawn by O.I.K.). Scale bar is 10 µm in a and 5 µm in e and h