Figure 2. IGF1R is a novel cargo of... | Rockefeller University Press

Figure 2.

$Figure 2. IGF1R is a novel cargo of KIF1Bβ. (A) Yeast two-hybrid assays assessing the direct binding between the ICD (961–1,373 aa) of mouse IGF1Rβ and various deletion mutants of mouse KIF1Bβ. Note that the 885–1,410-aa region within the KIF1Bβ stalk domain is the minimal essential region for its binding. PH, Pleckstrin homology. (B) Endogenous vesicle IP. IB of the brain lysates (Lys) of Kif1bβGFP/GFP and Kif1bβ+/+ (WT) mice and their immunoprecipitates using an anti-GFP antibody (IP by anti-GFP) labeled with the indicated antibodies. (C) Nycodenz gradient vesicle flotation assay of adult mouse brain lysates labeled with the indicated antibodies. Note that KIF1Bβ, IGF1Rα, IGF1Rβ, and synaptophysin are detected in the same fractions 14–15. (D) A Coomassie Brilliant Blue–stained gel for a pulldown assay between purified GST–KIF1Bβ885–1,410 and IGF1RβICD–3×FLAG showing their direct interaction. (E and F) Double-color time-lapse fluorescence images of a dissociated hippocampal neuron at DIV7 transfected with KIF1Bβ-EGFP and SP-IGF1Rβ-TagRFP using spinning-disk confocal microscopy equipped with 100×/1.46 Plan Apochromat oil-immersion objective lens (E) and its kymograph (F). The cell body is on the left side. Bars, 10 µm. Note that the speed of the comigrating vesicle (arrows in E) is ∼1 µm/s. See Video 2.$

IGF1R is a novel cargo of KIF1Bβ. (A) Yeast two-hybrid assays assessing the direct binding between the ICD (961–1,373 aa) of mouse IGF1Rβ and various deletion mutants of mouse KIF1Bβ. Note that the 885–1,410-aa region within the KIF1Bβ stalk domain is the minimal essential region for its binding. PH, Pleckstrin homology. (B) Endogenous vesicle IP. IB of the brain lysates (Lys) of Kif1bβ^GFP/GFP and Kif1bβ^+/+ (WT) mice and their immunoprecipitates using an anti-GFP antibody (IP by anti-GFP) labeled with the indicated antibodies. (C) Nycodenz gradient vesicle flotation assay of adult mouse brain lysates labeled with the indicated antibodies. Note that KIF1Bβ, IGF1Rα, IGF1Rβ, and synaptophysin are detected in the same fractions 14–15. (D) A Coomassie Brilliant Blue–stained gel for a pulldown assay between purified GST–KIF1Bβ885–1,410 and IGF1RβICD–3×FLAG showing their direct interaction. (E and F) Double-color time-lapse fluorescence images of a dissociated hippocampal neuron at DIV7 transfected with KIF1Bβ-EGFP and SP-IGF1Rβ-TagRFP using spinning-disk confocal microscopy equipped with 100×/1.46 Plan Apochromat oil-immersion objective lens (E) and its kymograph (F). The cell body is on the left side. Bars, 10 µm. Note that the speed of the comigrating vesicle (arrows in E) is ∼1 µm/s. See Video 2.