Fig 2: A2AR promotes Cbl-b-mediated Notch1 degradation via STS-1. Mass spectrometry analysis was carried out in lysates where Notch1 was immunoprecipitated from primary mouse CD8+ T-cells stimulated with anti-CD3/CD28 and treated with vehicle (control, DMSO) or 1µM ZM-241385 (ZM) or 1µM CGS-21680 (CGS) or ZM+CGS, as indicated in the figure. (A) Heat map of mass spectrometry analysis of Notch1 interactome. Uniprot IDs of the detected proteins are indicated on the right side of the heat map. Protein abundance is expressed as NSAF log2 fold-change on a color scale from blue to red where blue indicates the lowest and red the highest value, respectively. (B) Volcano plot of proteins identified in the mass spectrometry in ZM-treated vs. CGS-treated CD8+ T-cells. The X axis indicates fold change (log2) and Y axis the p-value (-log10). The dotted lines indicate the cut-off separating proteins with p<0.05 (blue and red dots) from non-significant ones (black dots). Proteins that are interacting the least with Notch1 are shifted to the left of the plot whereas the ones interacting the most are shifted to the right. The bottom graph shows the interaction between Notch1 and STS-1 (protein abundance) from the mass spectrometry analysis. (C) Protein levels of Notch1, STS-1 and Cbl-b in primary activated CD8+ T-cells isolated from C57BL6 (WT) or STS-1/2 -/- (STS-1/2 KO) mice and treated with vehicle (control, DMSO) or 1µM CGS. Densitometry (O.D.) results for N1TM and Cbl-b normalized by ß-actin are shown below the panel. (D) Top 10 ubiquitin ligases interacting with Notch1 ordered by the most to the least interacting based on mass spectrometry results (protein abundance). The graphs show averages ± standard deviation from three independent experiments. **p<0.01, two tailed T-test with equal variance.

Fig 3: PSGL-1 co-localizes with the TCR, Zap70, and the Zap70 inhibitor Sts-1(A) Immunofluorescence staining (40× magnification, scale bar of 10 µm) of CD3 (green) and PSGL-1 (red) on CD8+ TK-1 cells before or after crosslinking with CD3 and/or PSGL-1 antibodies. Experiments were performed 2×.(B) Chromatin accessibility within the Ubash3b gene region in WT OT-I cells (black) or PSGL-1-/- OT-I cells (red). Biological replicates prepared from 2 separate experiments.(C) Western blot of Sts-1 and ß-actin expression in naive (top) or 2 day activated (bottom) WT and PSGL-1-/- OT-I CD8+ T cells. Each band is a biologic replicate. Experiments were performed 2×.(D and E) Imaging flow cytometry analysis of Sts-1, PSGL-1, CD3, and Zap70 localization and co-localization in 10,000 naive and activated WT and PSGL-1-/- OT-I CD8+ T cells.(D) Similarity scores of Sts-1, Zap70, and PSGL-1 expression in naive (left) or 20 min activated (right) WT (gray/black) and PSGL-1-/- (light red, red) OT-I cells. Error bars are median absolute deviation (MAD).(E) Representative images from (D) in two WT OT-I CD8+ T cells.Experiment performed 2×. See also Figures S3 and S4.