Fig. 7

Identification of Klhl13 target proteins that mediate its effect on pluripotency and differentiation. a Schematic representation of the putative mechanism underlying Klhl13’s (blue) pluripotency-promoting effects, where differentiation-promoting substrate proteins (red) are targeted for proteasomal degradation through recruitment to the Cul3 E3 ubiquitin ligase complex via Klhl13’s Kelch domain. b Experimental strategy for the identification of Klhl13 target proteins: Putative substrates should be more abundant at the protein level in Klhl13-deficient cells and should interact with Klhl13 and with the Kelch domain only. To identify substrates, the proteomes in wildtype and K13-HOM cells were compared and Klhl13/Kelch interaction partners were identified through GFP-mediated IP-MS. All three datasets were integrated to identify candidate proteins. c, d Volcano plots of the IP-MS results for the GFP-Kelch (c) and GFP-Klhl13 (d) constructs. The mean fold-change across 3 biological replicates relative to the GFP-only control against the p value calculated via a two-sample Student’s t test with Benjamini-Hochberg correction for multiple testing is shown. Black lines indicate the significance threshold that was chosen such that FDR < 0.1, assuming that all depleted proteins (left-sided outliers) were false-positive. Triangles show known Klhl13 interaction partners extracted from the BioGRID. e Volcano plot showing the proteome comparison of 1.8XX wildtype cells and K13-HOM mESCs. The mean fold-change across 3 biological replicates is shown. Proteins that are upregulated upon Klhl13 depletion are highlighted in red (p < 0.05 of Student’s t test, Benjamini-Hochberg FDR). Circles in c–e depict putative Klhl13 substrate proteins that were found to interact with Klhl13 and the Kelch domain and were upregulated upon Klhl13 deletion. f Venn diagram summarizing results in c–e. g Model of how X-dosage modulates the MAPK signaling pathway, pluripotency factor expression, and differentiation