Extended Data Fig. 5: Enhanced recycling and insensitivity to degradation in CTLA-4 Kless cells.

a). CHO cells expressing WT or CTLA-4 lacking cytoplasmic lysine residues (Kless) were stained for CTLA-4 recycling using anti-CTLA-4 antibodies at 37 °C and detected by flow cytometry. The percentage of CTLA-4 recycled is shown in the presence and absence of MG132 to assess the impact of ubiquitylation. b). Flow cytometry analysis of WT and Kless cells stained for CTLA-4 at 4°C (to stain cell surface), 37 °C (to stain cycling) and following fixation and permeabilisation (to stain total) in the presence or absence of MG132. c). Impact of Kless mutation knocked in to the CTLA-4 locus in activated human CD4+ T cells using CRISPR-Cas9/AAV6 HDR. Cycling CTLA-4 was detected using anti-CTLA-4 antibody at 37°C for 1h in cells receiving either a WT or Kless CTLA-4 repair template (histograms). The amount of cycling CTLA-4 was compared between edited (GFP⁺) and non-edited (GFP⁻) cells in the same culture. Data shown is collated from 3 biologically independent samples. Statistical significance was determined by 2-way ANOVA with Sidak’s multiple comparison correction, ****P < 0.0001. All data are presented as mean ± s.d. and show individual data points from 3 biologically independent samples.