Fig. 4: Polyubiquitination is required for SERINC5 localization to the plasma membrane.

A Ser5 was expressed with Cas9, indicated proteins, and indicated sgRNAs in HEK293T cells. Ser5 expression was compared by WB. B Ser5-EGFP was expressed in HeLa and Jurkat cells in the presence of KLHL20- or Cul3-KD by CRISPR/Cas9. KLHL20 and Cul3 in these KD cells were also complemented by their ectopic expression. After staining with DAPI, Ser5 subcellular localization was detected by confocal microscopy (scale bar 2 or 5 µm). Experiments were repeated twice, and representative experiments are shown. C Ser5-iFLAG was expressed in HEK293T and Jurkat cells in the presence of ectopic KLHL20 and Cul3 expression and/or their KDs by CRISPR/Cas9. Cells were stained with anti-FLAG and levels of Ser5 on the cell surface were analyzed by flow cytometry. Results are shown as relative values, with the surface expression of Ser5 alone set as 100. Error bars indicate SEMs calculated from two or three experiments. n = 3 (HEK293T) or n = 2 (Jurkat); One-way ANOVA Tukey test; ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001. D Ser5 was expressed in HEK293T cells in the presence of ectopic KLHL20 and Cul3 expression and/or their KDs by CRISPR/Cas9. The plasma membranes from these cells were purified and analyzed by WB. E Ub-VN was expressed with Ser5-VC or its lysine mutants in HeLa cells. Cells were stained with DAPI and anti-FLAG for Ser5. BiFC fluorescent signals were detected by confocal microscopy (scale bar 2 or 5 µm). Experiments were repeated twice, and representative experiments are shown. Source data are provided as a Source Data file.