kif13a knockdown constructs  (OriGene)

Journal: Life Science Alliance

Article Title: Axonal transport of Hrs is activity dependent and facilitates synaptic vesicle protein degradation

doi: 10.26508/lsa.202000745

Figure Lengend Snippet: (A) Schematic diagram of transport assay using GFP/FKBP-tagged BicD2 motor domain, Myc/FRB-tagged kinesin cargo-binding tail domain, and fluorescently tagged cargo of interest. Upon addition of the rapamycin analogue (rapalog), FKBP and FRB interact and are co-transported to the centriole. If the protein/vesicle of interest interacts with the kinesin tail, it is also transported. (B, C) Images of N2a cells expressing RFP-Hrs (red), BicD2-GFP-FKBP (green), and KIF13A-FRB-Myc (blue), +/− rapamycin analogue to induce FKBP/FRB interaction (linker) (B), and corresponding line scan graphs (taken from left to right along the yellow lines in merged images; distance expressed in pixels) (C), showing that addition of linker induces colocalization of Hrs with BicD2 and KIF13A as depicted by a single peak in the lower graph. Size bar, 10 μm. (D, E) Images of N2a cells expressing RFP-Hrs (red), BicD2-GFP-FKBP (green), and KIF13B-FRB-Myc (blue), +/− linker (D), and corresponding line scan graphs (taken along yellow lines in merged images) (E) showing that addition of linker does not induce Hrs colocalization with BicD2/KIF13B. Size bar, 10 μm. (F) Percentage of transfected cells exhibiting rapalog-induced Hrs recruitment to the centriole (* P = 0.0178, unpaired t test, n = 4 fields of view/condition). (G) Immunoblot of lysates from N2a cells coexpressing mCherry or RFP-Hrs together with KIF13A-FRB-Myc or KIF13B-FRB-Myc, immunoprecipitated (IP) with mCherry antibody and probed with Myc or mCherry antibodies. (H) Quantitative analysis of co-immunoprecipitated KIF13A/B, normalized to immunoprecipitated RFP-Hrs, and expressed as intensity normalized to KIF13B (**** P < 0.0001, unpaired t test, n = 5 independent experiments). Bars show mean ± SEM.

Article Snippet: Full-length [Chmp4b] shRNA scrambled control and KIF13A knockdown constructs (shCtrl, shKIF13A1 and A2, KIF13B) were purchased from Origene (Cat. no. TL706020).

Techniques: Transport Assay, Binding Assay, Expressing, Transfection, Western Blot, Immunoprecipitation

Journal: Life Science Alliance

Article Title: Axonal transport of Hrs is activity dependent and facilitates synaptic vesicle protein degradation

doi: 10.26508/lsa.202000745

Figure Lengend Snippet: (A, B) Images of N2a cells coexpressing BicD2-GFP-FKBP (green) and KIF13A-FRB-Myc (blue; A) or KIF13B-FRB-Myc (blue; B), +/− rapamycin analogue to induce FKBP/FRB binding (linker). Size bar, 10 μm. (C, D) Images of N2a cells expressing mCh-Rab5 (red), BicD2-GFP-FKBP (green), and KIF13A-FRB-Myc (blue), +/− linker, and corresponding line scan graphs (taken along yellow lines in merged images) (D) showing that addition of linker induces colocalization of Rab5 with BicD2/KIF13A. (E, F) Images of N2a cells expressing mCh-Rab5 (red), BicD2-GFP-FKBP (green), and KIF13B-FRB-Myc (blue), +/− linker, and corresponding line scan graphs (taken along yellow lines in merged images) (F) showing that addition of linker induces colocalization of Rab5 with BicD2/KIF13B.

Article Snippet: Full-length [Chmp4b] shRNA scrambled control and KIF13A knockdown constructs (shCtrl, shKIF13A1 and A2, KIF13B) were purchased from Origene (Cat. no. TL706020).

Techniques: Binding Assay, Expressing

Journal: Life Science Alliance

Article Title: Axonal transport of Hrs is activity dependent and facilitates synaptic vesicle protein degradation

doi: 10.26508/lsa.202000745

Figure Lengend Snippet: (A) Immunoblot of lysates from 13 to 15 day in vitro (DIV) hippocampal neurons expressing shKIF13A1, shKIF13A2, or scrambled shRNA control (shCtrl), probed with antibodies against KIF13A and tubulin. (B) Quantification of KIF13A band intensity, normalized to tubulin control, with shCtrl or shKIF13A expression (*** P = 0.0004 [shKIF13A1], *** P = 0.0002 [shKIF13A2], one-way ANOVA with Dunnett’s multiple comparisons test; n = 4 independent experiments). Bars show mean ± SEM. (C) Images of axons from 14 DIV dissociated hippocampal neurons expressing shCtrl or shKIF13A1, immunostained for neurofilament and KIF13A. (D) Quantification of KIF13A puncta per 100 μm axon (** P = 0.0012, unpaired t test, n = 18 [shCtrl] and 15 [shKIF13A1] fields of view/condition). (E) Representative immunoblot of lysates from 13 to 15 DIV hippocampal neurons expressing shKIF13B or scrambled shRNA control (shCtrl), probed with antibodies against KIF13B and tubulin. (F) Quantification of KIF13B band intensity, normalized to tubulin control, with shCtrl or shKIF13B expression (*** P = 0.0007, unpaired t test; n = 4 independent experiments). Bars show mean ± SEM. (G) Images and corresponding kymographs of EGFP-Hrs in axons from dissociated hippocampal neurons expressing shCtrl (left panels), shKIF13A2 (middle panels), or shKIF13B (right panels), under control conditions (upper panels) and after 2 h treatment with Bic/4AP (lower panels). Horizontal size bar, 10 μm, vertical size bar, 25 s. One frame taken every 5 s (0.2 fps). Dashed yellow lines highlight axons. Quantification of Hrs motility under these conditions is shown in . (H) Images and corresponding kymographs of EGFP-Hrs in axons from dissociated hippocampal neurons expressing shCtrl (left panels) or shKIF13A1 (right panels), under control conditions (upper panels) and after 1 h treatment with BDNF (lower panels). Horizontal size bar, 10 μm, vertical size bar, 25 s. One frame taken every 5 s (0.2 fps). (I) Percentage of motile Hrs puncta in axons (* P = 0.0298, ns P = 0.9912, one-way ANOVA with Tukey’s multiple comparisons test; n = 24 [shCtrl, control], 25 [shCtrl, BNDF], 20 [shKIF13A1, control], and 23 [shKIF13A1, BDNF] videos/condition).

Article Snippet: Full-length [Chmp4b] shRNA scrambled control and KIF13A knockdown constructs (shCtrl, shKIF13A1 and A2, KIF13B) were purchased from Origene (Cat. no. TL706020).

Techniques: Western Blot, In Vitro, Expressing, shRNA, Control

Journal: Life Science Alliance

Article Title: Axonal transport of Hrs is activity dependent and facilitates synaptic vesicle protein degradation

doi: 10.26508/lsa.202000745

Figure Lengend Snippet: (A, B) Images of proximity ligation assay (PLA) in 13–15 day in vitro dissociated hippocampal neurons expressing EGFP (A) or EGFP-Hrs (B) under control or 2 h Bic/4AP conditions. PLA puncta (red), representing EGFP-Hrs/KIF13A interactions, were quantified in the soma (dashed yellow ovals). Size bar, 10 μm. (C) PLA puncta number per soma (* P = 0.0129, **** P < 0.0001, one-way ANOVA with Sidak’s multiple comparisons test; n = 13 [EGFP control], 9 [EGFP-Hrs] condition, ≥3 independent experiments). (D) Immunoblot of lysates from N2a cells coexpressing RFP-Hrs and KIF13A-FRB-Myc, treated with control or high K+ Tyrodes solution for 2 h, immunoprecipitated (IP) with mCherry antibody and probed with Myc or mCherry antibodies. (E) Quantitative analysis of co-immunoprecipitated KIF13A, normalized to immunoprecipitated RFP-Hrs and expressed as intensity normalized to control condition (* P = 0.0249, unpaired t test, four independent experiments). Bars show mean ± SEM.

Article Snippet: Full-length [Chmp4b] shRNA scrambled control and KIF13A knockdown constructs (shCtrl, shKIF13A1 and A2, KIF13B) were purchased from Origene (Cat. no. TL706020).

Techniques: Proximity Ligation Assay, In Vitro, Expressing, Control, Western Blot, Immunoprecipitation

Journal: Life Science Alliance

Article Title: Axonal transport of Hrs is activity dependent and facilitates synaptic vesicle protein degradation

doi: 10.26508/lsa.202000745

Figure Lengend Snippet: Neuronal firing increases the association of Hrs with plus-end directed kinesin motor protein KIF13A, stimulating the anterograde transport of these vesicles and their delivery to synaptic vesicle pools. Hrs+ vesicles also transport STAM1 and are a subset of Rab5+ early endosomes.

Article Snippet: Full-length [Chmp4b] shRNA scrambled control and KIF13A knockdown constructs (shCtrl, shKIF13A1 and A2, KIF13B) were purchased from Origene (Cat. no. TL706020).

Techniques: