Fig. 4. Ribosome assembly factor PWP1 is a substrate of CRL4^DCAF1 E3 ubiquitin ligase.

(A) 3xFLAG knock-in (KI) to endogenous DCAF1 in HeLa cells verified by genomic PCR and immunoblotting. WT, wild type; WB, Western blot. (B) Immunopurification of two endogenous DCAF1 complexes from HeLa cells. Molar excess competing antigen peptides added in control immunocomplexes. (C) Scatter plot of log₂ ratios of 626 proteins identified by mass spectrometry analyses of two DCAF1 immunocomplexes. (D) Venn diagram of proteins with greater than fourfold abundance change. (E) List of 17 DCAF1-interacting proteins identified in both DCAF1 immunocomplexes. (F) Interaction between ectopically expressed PWP1 and DCAF1 in U2OS cells by coimmunoprecipitation (co-IP). (G) Endogenous interaction between DCAF1 and PWP1 in HeLa cells treated with 20 μM MG132 for 6 hours. (H) Dcaf1^f/−;Cre-ERT2 MEFs with 200 nM 4OHT or dimethyl sulfoxide (DMSO) for 2 days. Protein-protein interactions by co-IP. (I) MEFs with 200 nM 4OHT for indicated days. (J) HeLa cells transfected with small interfering RNAs (siRNAs) targeting indicated genes individually. (K) HeLa cells stably expressing HA-tagged ubiquitin transfected with siRNAs targeting indicated genes and treated with MG132. Endogenous PWP1 was immunoprecipitated and immunoblotted. (L) Immunopurified PWP1 protein incubated with CRL4^DCAF1 E3 immune complex in the presence or absence of UBA1 (E1), UBCH5C (E2), adenosine triphosphate (ATP), and ubiquitin in vitro for 1 hour. Ubiquitylated PWP1 was immunoprecipitated and immunoblotted.