Supplementary Figure 5: Model predicting degradative or non-degradative ubiquitylation is assessed by immunoblot.

a, Ubiquitylation, WCP, and RNA-seq expression changes for all proteins exhibiting TCR-induced ubiquitylation ( > 25% increase) in CD4 + T cells unstimulated or stimulated for 4 h with CD3 + CD28 antibody coated beads (3:1 cell:bead ratio). Circle size corresponds to increase in ubiquitylation normalized log₂ fold change. Proteins exhibiting consistent protein abundance and increased RNA are predicted to be degraded by ubiquitin (gray) while the remaining proteins are predicted to be non-degradative outcomes of ubiquitylation (red). Translucent blue filled circles indicate those proteins were tested for experimental validation of the prediction method. b, Western blot showing protein abundance of 7 selected proteins, from those described in (a), for CD4 + T cells unstimulated or stimulated with CD3 + CD28 antibody (plate-bound antibody, 5 μg/mL), for the indicated time-course. Cycloheximide (CHX) was added after 1 h of stimulation CD3 + CD28 antibody stimulation. Comparing protein levels at 1 h (no CHX) to 4 h with CHX added at 1 h suggests that LAT, MYCBP2 and SIN3B are significantly decreased, while GRAP, PKCθ, SNX18 and ZAP-70 remain stable. LC indicates loading control. Representative blots from three independent experiments are shown. c, Quantification of normalized intensity changes calculated for 4 h TCR stimulation with CHX added after 1 h compared to the protein intensity at 1 h, as described in (b). Decreases in protein levels with the addition of CHX (delta intensity < 1) for LAT, MYCBP2 and SIN3B indicate that these proteins are significantly decreased in abundance. Protein band intensity was normalized to corresponding loading control intensity. Mean fold changes ± sd of the three biological replicates are shown. Fold change of 1 indicates no change in abundance. Statistics were calculated using two-tailed, unpaired t-tests.