KLHL17/AF knockdown impairs functional synapse formation. Cultured hippocampal neurons were transfected with the indicated plasmids at 12 DIV and harvested for immunostaining using PSD95 (a, c–e) and SVP38 (b, f–h) antibodies at 18 DIV. Super-resolution images of AF distribution and that of synaptic proteins PSD95 (a) and SVP38 (b) in mature neurons, including whole cells and enlarged dendritic protrusions. c–h AF knockdown reduces functional synapse formation. c, f Representative confocal images, including whole cells and enlarged dendrite segments. d, g Representative confocal images of dendritic protrusions and heat maps. e, h Quantification of e PSD95- or h SVP38-positive protrusions and relative signal intensities. Samples were randomly collected from two independent experiments without knowing the treatment. N, the number of analyzed dendrites; n, the number of analyzed dendritic protrusions. Data represent the mean plus SEM. **P < 0.01; ***P < 0.001; ns, not significant. Scale bars: a, b whole cell: 10 μm, enlarged inset: 1 μm; c, f whole cell: 20 μm, enlarged inset: 2 μm; d, g 1 μm. One-way ANOVA (e, h).