Extended Data Fig. 3. RASA2 is a TCR stimulation-dependent attenuator of Ras-MAPK signaling.
a, Western blot showing effect of RASA2 ablation on the level of active Ras in primary human T cells with or without TCR stimulation. b, Densitometry measurements calculated for p-MEK and p-ERK westerns and averaged for all 3 T cell donors (mean ± SEM, *p < 0.05 and **p < 0.01 for two-sided paired t-test) c, Western blots showing the effect of RASA2 ablation compared to control-edited T cells for phospho-MEK and phospho-ERK over time after TCR stimulation in primary human T cells from 3 human donors from (b). d, Flow cytometry plots showing representative gating for phospho-proteins in the Ras signaling pathway in 2 human donor T cells after TCR stimulation. e, Summary of MFI for phospho-proteins in Ras signaling pathways over time after TCR stimulation. Y-axis shows MFI for each marker divided by MFI for FSC-A to normalize for cell size (mean +/− SEM, n = 2 donors in 3 replicates, **p < 0.01 for two-sided Wilcoxon test). f, T cells from 2 donors after 13 days of expansion in culture split into cultures with or without IL2 in triplicates and viability was tracked over time. Lines are mean, individual dots are replicates. g, Flow cytometry histogram plots from stimulated and unstimulated T cells for pERK, CD69 and CFSE. Baseline levels of RASA2 KO and control-edited T cells remained similar (except for variability in CD69 levels), while after CD3/CD28 stimulation, RASA2 KO T cells showed higher levels of pERK, CD69 and proliferation over CTRL KO T cells. Results representative of 4 human donors. We noted heterogeneous expression of CD69 at baseline across donors, with some showing marginally higher levels in RASA2 KO T cells.