Extended Data Fig. 2: FBXO38 specifically regulates PD-1 protein level.
From: FBXO38 mediates PD-1 ubiquitination and regulates anti-tumour immunity of T cells
a, Generation of FBXO38-HA knock-in and FBXO38 knockout Jurkat T cells by CRISPR–Cas9 technology. The knock-in and knockout efficiencies were confirmed by immunoblotting. b, Co-immunoprecipitation of Myc–FBXO47 and HA–PD-1 in HEK293FT cells. WCL, whole-cell lysate. c–f, Effect of FBXO38 and FBXO47 overexpression on surface PD-1 levels in Jurkat T cells. Cells were transduced by Myc-FBXO38-IRES-GFP, Myc-FBXO47-IRES-GFP or vector control using lentiviruses. Transduced cells were sorted by GFP expression. c, FBXO38 or FBXO47 protein levels. d, Surface TCR levels in unstimulated Jurkat cells were measured by flow cytometry. e, f, Cell-surface levels of PD-1 in Jurkat cells stimulated with anti-CD3 (5 μg ml−1) for 24 h. n = 3 biologically independent samples. g–m, Effect of FBXO38 knockdown on PD-1 expression levels in Jurkat T cells. Cells were transduced with constructs containing control shRNA or FBXO38 shRNA (knockdown) as well as a GFP marker. Transduced cells were sorted by GFP expression. g, h, FBXO38 expression and transcriptional level in control and knockdown cells. i, Surface TCR levels in unstimulated Jurkat cells. j, Total PD-1 levels in control and knockdown cells stimulated with PHA (150 ng ml−1) for 72 h. k–m, Surface PD-1 levels in control and knockdown cells stimulated with PHA (50, 75 or 100 ng ml−1) for the indicated number of hours. n = 3 biologically independent samples. n–p, Effect of Fbxo38 overexpression on PD-1 surface levels in activated OT-1 CD8+ T cells. Activated OT-1 T cells were transduced by Myc-FBXO38-IRES-GFP or vector control using a retrovirus. PD-1 levels in transduced CD8+ T cells (GFP positive) were measured 24 and 48 h after transduction. n, Transduction efficiency in activated OT-1 T cells. o, p, PD-1 surface levels in vector control and Fbxo38-overexpressing (FBXO38) OT-1 T cells. n = 3 biologically independent samples. Data are representative of two (j) or three (a–i, k–p) independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired Student’s t-test except the 72-h group of PD-1 MFI in m, in which an unpaired Student’s t-test with Welch’s correction was used. For gel source data, see Supplementary Fig. 1.
