Fig. 1. Identification of Cul3-KLHL20 as a SERINC5 ubiquitin E3 ligase via mass spectrometry.
A A list of E3 Ub ligases that were identified from the SERINC5 protein complex by mass spectrometry. B Ser5 was expressed with Ub in the presence of Skp1and/or Cul1 in HEK293T cells. Similarly, Ser5 and its 1-19 K/R mutant were expressed with Ub in the presence of KLHL20 and/or Cul3. Expression of these proteins was analyzed by WB using anti-epitope tag antibodies. C Cas9 was expressed with Cul3- or KLHL20-specific sgRNAs in HEK293T cells. Expression of Cul3 and KLHL20 was determined by WB using their specific antibodies. D Ser5 was expressed with Cas9, GFP, KLHL20, Cul3, and/or KLHL20- or Cul3-sgRNAs in HEK293T cells. Ser5 was immunoprecipitated by anti-FLAG, and levels of GFP, KLHL20, and Cul3 in cell lysate (Input) and IP samples were compared by WB. E Ser5 was expressed with KLHL20 and its ∆K and 5A mutants in HEK293T cells. Ser5 was immunoprecipitated and levels of these different KLHL20 proteins in cell lysate and IP samples were compared by WB. F Ser5 was expressed with Ub mutant UbK48/63R, KLHL20, Cul3, and/or Cas9 plus KLHL20- or Cul3-sgRNAs in HEK293T cells. Ectopic Ub was immunoprecipitated by anti-HA and levels of polyubiquitinated Ser5 were compared by WB. G Ser5 was expressed with Ub, KLHL20 or its mutants ∆K and 5A, and Rbx1 in HEK293T cells. Ectopic Ub was immunoprecipitated by anti-HA and levels of polyubiquitinated Ser5 were compared by WB. Ser5, serine incorporator 5; Cul, cullin; Ub, ubiquitin; Skp1, S-phase kinase-associated protein 1; KLHL20, Kelch-like protein 20; Cas9, CRISPR associated protein 9; sg, short guide RNA; GFP, green fluorescent protein; Rbx1, RING-box protein 1; ∆K, KLHL20 mutant with Kelch-repeat domain deleted; 5A, KLHL20 mutant with V110, R112, I114, L147, and Q149 mutated to alanine; HA, hemagglutinin-tag; His, polyhistidine-tag; FLAG, FLAG-tag. All experiments were repeated at least twice, and similar results were obtained. Source data are provided as a Source Data file.