Fig 1: PAG is enriched at the immune synapse(A) PAG-GFP was overexpressed in A549 cells prior to cell lysis and GFP immunoprecipitation. Mass spectrometry (MS) was then performed and identified that the extracellular portion of PAG was intact and uncleaved. Purple text indicates peptides detected by MS, yellow highlight shows the transmembrane domain, and blue highlight specifies phosphorylated tyrosines. (B) Predicted structure of the extracellular portion of human and mouse PAG. (C) Western blot of Jurkat T cells following plate-bound stimulation. PAG (80 kDa) is phosphorylated PAG on the basis of size; immunoblot PAG (clone MEM-255). (D) Proximity ligation assay of Jurkat T cells incubated with control or PD-L2-overexpressing Raji B cells in the presence of SEE. Cells were permeabilized, and the proximity between endogenous PD-1 and PAG was imaged using intracellular antibodies. Clusters indicate a proximity of ≤40 nm. Quantification is done as number of clusters per cell over 2 independent experiments, with 1,400–2,000 cells counted per condition over the two experiments; graphed points represent fields of cells. ∗∗∗p ≤ 0.001. (E) Schematic representation of full-length PAG-GFP- and Fc-conjugated PAG-GFP. (F) Jurkat T cells were transfected with PAG-GFP or Fc-PAG-GFP and incubated with Raji B cells in the presence of SEE and cells were imaged using confocal microscopy to assess the location of PAG relative to the immune synapse. (G) Quantification of the number of cells per phenotype. Percentages were counted for 5 independent experiments. ∗∗∗∗p ≤ 0.0001. (H) Jurkat T cells were transfected with PAG-GFP or Fc-PAG-GFP and incubated with Raji B cells in the presence of SEE. IL-2 secretion was measured using ELISA. n = 2; ∗p ≤ 0.05.
Fig 2: Clone 7M16A binds to the extracellular domain of PAG(A) ELISA with immobilized PAG peptide (human, mouse, or cynomolgus) using hybridoma supernatants. (B) 7M16A staining of non-permeabilized wild-type C57BL6 or PAG knockout (KO) murine splenocytes shows specificity of binding. (C) Western blot of A549 cells made to stably overexpress PAG-GFP. (D) Western blot of fractionated cell lysates showing that PAG-GFP is expressed at high levels at the cell membrane. Confocal imaging showing the localization of PAG-GFP at the plasma membrane. (E) 7M16A staining of non-permeabilized A549 cells shows binding to PAG-GFP on the surface. (F) ELISA with immobilized human PAG peptide using purified monoclonal antibodies. (G) ELISA of IL-2 secretion from primary human CD3+ T cells stimulated as indicated for 48 h. n = 3; ∗p ≤ 0.05 and ∗∗p ≤ 0.01. (H and I) Jurkat T cells stably express PAG-GFP with or without PD-1-SNAP incubated with Raji B cells in the presence of SEE. Cells were imaged using confocal microscopy to assess the location of PAG and PD-1 relative to the immune synapse. White arrow indicates the center of the immune synapse where PAG was excluded with 7M16A pretreatment. White star indicates the point in the cell with enrichment of PD-1 either at or away from the immune synapse. Quantification of the number of cells per phenotype. ∗p ≤ 0.05.
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