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. 2023 Apr 3;12(4):1081–1093. doi: 10.1021/acssynbio.2c00587

Figure 4.

Figure 4

Immune checkpoint REULR. (A) Schematic representation of PD1-REULR-mediated PD-1 degradation. (B–D) HEK293T cells were transiently transfected with FLAG-tagged full-length PD-1 cDNA (human) under the control of a constitutively active CMV (cytomegalovirus) promoter. 24 h post-transfection, cells were incubated with PD1-REULR molecules (50 nM), as indicated using RNF128 (E1; E2)-, RNF130 (A1; D1)-, or RNF167 (A5)-targeting nanobodies fused to a PD-1 binding nanobody (PD1). Monomeric binding moieties or PBS were used as negative controls. After 24 h, cells were subjected to FACS analysis using a FLAG antibody (Alexa Fluor 647 conjugate) to monitor PD-1 levels on the cell surface. Representative FACS histograms are visualized below the quantified data. Data are mean ± s.d. (n = three replicates).