Fig. 3: Kif5b plays a role in CCV coating without affecting cell peripheral distribution of CCV coat proteins and uncoating catalyst.

a Representative immunofluorescence staining images for CHC, α-Adaptin, and Hsc70 in shKif5b or shControl RNA treated Neuro2a cells. Green dashed lines delineate the cell surface defined by co-staining of F-actin with phalloidion. Scale bar = 10 μm. b Quantitative analysis of subcellular distribution of CHC, α-Adaptin, and Hsc70. The distribution was quantified by measuring the line profile of fluorescence intensity from the plasma membrane (PM) to the nucleus (See also Supplementary Fig. S6). Error bars indicate s.e.m. n = 30. ns, not significant (P > 0.05). c, d Representative Western blots of cortical proteins or uncoating-related proteins in cortices of kif5b mutant mice or control littermates. e Left column: immunoprecipitation of Hsc70 in kif5b mutant mouse cortices or controls. The precipitated Hsc70, co-precipitated CHC or input cortical lysates were subjected to Western blot analysis. Right column: quantitative analysis of bands of precipitated Hsc70 and co-precipitated CHC from mutant cortices (n = 4) and controls (n = 4). Error bars indicate s.e.m. **P < 0.01. f left column: uncoating assay of CCVs from mutant or control mouse cortices. The amount of CHC released into supernatants was representative of uncoating. Right column: quantitative analysis of released CHC. Error bars indicate s.e.m. n = 3. *P < 0.05. g, h Uncoating assay of CCVs treated with or without different recombinant fragments of Kif5b. i Representative disassembly curve of purified cortical CCVs from mutant or control mice detected by light scattering assay in vitro