Supplementary Figure 11: PUP-IT^IL2-dependent cell-surface modification of bio-DE28.

(a) Activated T cell surface can be modified by PUP-IT^IL2. T cells are first activated then mixed with PUP-IT^IL2, bio-DE28, ATP and rapamycin. The same reaction was also set up without either ATP or rapamycin. A complete assembly of PUP-IT^IL2 is required for bio-DE28 labeling. This experiment was repeated independently more than three times with similar results. (b) Cell surface labeling increases with increased PUP-IT^IL2. 1 × 10⁵ Jurkat cells were first activated over night by anti-CD3 and anti-CD28. Cell surface Pup modification assays were set up the same as in Fig. 6. In 150 μl reaction volume, IL2-FKBP was added to reach the final concentration at 0.5, 1, 2, 4, and 14.8 ng/μl. With increased IL2-FKBP protein, more cell surface labeling of bio-DE28 can be observed. This experiment was repeated independently twice with similar results. (c) Representative flow cytometer data from three experimental repeats as shown in Fig. 6d. (d) The gating strategy used in (a)-(c).