Fig. 5. NEK9 is a selective autophagy adaptor for MYH9.

a Results of mass spectrometry analysis of FLAG-NEK9 or FLAG immunoprecipitates. The x- and y-axes represent Peptide Spectrum Match (PSM) and abundance ratio (FLAG-NEK9 / FLAG), respectively. Proteins with an abundance ratio above five were tested for actual interaction with NEK9. Proteins above the dotted line were detected only in FLAG-NEK9 immunoprecipitates. See also Supplementary Data 2. b Immunoprecipitation using MEFs stably expressing FLAG or FLAG-NEK9 after serum starvation (4 h). Data are representative of three independent experiments. c Immunofluorescence microscopy of wild-type and Nek9^W967A MEFs stably expressing GFP-MYH9 after serum-starvation (4 h). Cells were stained with anti-NEK9 and anti-LC3 antibodies. Scale bars, 10 µm and 3 µm (insets). d Quantification of the number of GFP-MYH9 puncta in (c). e Colocalization between GFP-MYH9 and endogenous LC3 in (c) was determined by calculating Pearson’s correlation coefficient between intensities within each cell. Data were collected from 100 cells for each cell-type in (d, e). Solid bars indicate the medians, boxes the interquartile range (25th–75th percentile), and whiskers the 10th to 90th percentile. f Immunoblotting of wild-type and Nek9^W967A MEFs. g Quantification of the intensity of the MYH9 bands in (f). Data represent the mean ± SEM of three independent experiments. h Immunoblotting of the indicated organs of five-month-old wild-type (WT) and Nek9^W967A/W967A mice (KI). Asterisks (*) indicate non-specific bands in skeletal muscles. Data are representative of three biologically independent replicates; p values correspond to Tukey’s multiple comparisons tests.