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Raw MS data were processed with MaxQuant software (v1.6.0.1) and searched against the mouse proteome database UniProtKB with 22,286 entries, released in December 2018. Parameters of MaxQuant database searching were a false discovery rate (FDR) of 0.01 for proteins and peptides, a minimum peptide length of seven amino acids, a first search mass tolerance for peptides of 20 ppm, and a main search tolerance of 4.5 ppm, and using the function “match between runs”. A maximum of two missed cleavages was allowed for the tryptic digest. Cysteine carbamidomethylation was set as fixed modification, while N-terminal acetylation and methionine oxidation were set as variable modifications. Contaminants, as well as proteins identified by site modification and proteins derived from the reversed part of the decoy database, were strictly excluded from further analysis.

Comparison of protein abundance for both the proteomics (K13-HOM vs XX wildtype) and the IP datasets (GFP-Kelch vs GFP, D-GFP-Klhl13 vs D-GFP) was performed with Perseus (v1.6.1.3). LFQ intensities, originating from at least two different peptides per protein group, were transformed by log2. Only groups with valid values in at least one group were used, and missing values were replaced by values from the normal distribution. Statistical analysis for differential expression was done by a two-sample t-test with Benjamini-Hochberg (BH, FDR of 0.05) correction for multiple testing. The processed output files can be found in Additional file 7: Table S6 (IP-MS) and Additional file 8: Table S7 (Proteome).

For the identification of Klhl13 interaction partners, cut-offs were set from the data displayed in the volcano plots using a previously published method [115]. Briefly, a graphical formula as a smooth combination of the following parameters was implemented:

x: enrichment factor of a protein

p: p value of the t-test, calculated from replicates

xo: fixed minimum enrichment

c: curvature parameter

We optimized parameters c and xo such as to have 10% FDR (left-sided outliers) while maximizing the number of right-sided outliers. In the case of the GFP-Kelch IP, c = 0.32 and xo = 0.02. For the D-GFP-Klhl13 IP, c = 0.28 and xo = 0.04. Proteins without an associated gene name were filtered out in further analyses. Known Klhl13 interaction partners were extracted from the Biogrid database (Arih1, Aurkb, C1qbp, Cd2ap, Cops2, Cops4, Cops5, Cops6, Cops7a, Cul3, Dcun1d1, Hsp90aa1, Kiaa1429, Klhl21, Klhl22, Klhl9, Mad2l1, Nhlrc2, Nudcd3, Tfg, Ube2m, Ubxn7, Usp11, Zmym4).

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