Fig. 7

Both AF-N and AF-C are required for KLHL17/AF to regulate F-actin remodeling. a Schematic of AF-N and AF-C truncated mutants. AF-N consists of amino acid residues 1-300, which includes the BTB domain. AF-C comprises the C-terminal amino acid residues 301-640, which covers the six Kelch domains. All AF, AF-N and AF-C constructs were tagged with a Myc cassette at their N-terminal end for detection. b Actin-binding ability of AF variants. Neuro-2A cells were cotransfected with GFP-actin and different AF constructs, as indicated, and subjected to immunoprecipitation and immunoblotting using the indicated antibodies. Since protein levels of full-length AF and AF-C were very low in Neuro-2A lysates, they were barely detectable in 2% input lysate. However, all AF variants could be enriched after immunoprecipitation using Myc antibody. Note that even though AF-N was expressed at much higher levels in Neuro-2A cells, only full-length AF and AF-C were able to precipitate actin. c–e Both AF-N and AF-C alter F-actin reorganization ability. COS1 cells were cotransfected with GFP and AF variants, as indicated, and treated with Latrunculin A (LatA, 1 µM) for 30 min to disrupt F-actin filament. After treatment, cells were recovered for a further 60 min and subjected to immunostaining using Myc tag antibody to label AF expression and by phalloidin staining to label F-actin. c Representative images. GFP images are shown in insets to indicate transfected cells. d Expression patterns and levels of AF variants in COS1 cells. e Quantification of cell area before and after LatA treatment. Samples were randomly collected from two independent experiments without knowing the treatment. N the number of analyzed cells. Data represent the mean plus SEM. ***P < 0.001; ns non-significant. Scale bar: c 20 μm, d 10 μm. One-way ANOVA (d, e)