(A–F) His 212 was mutated into neutrally charged residues (His 212 Ala, His 212 Tyr), or into positively charged residues (His 212 Arg, His 212 Lys). RasGRP1 signaling was determined by levels of phosphorylated ERK (P-ERK) and S6 (P–S6). Graphs depict fold changes of P-ERK or P-S6 relative to Wildtype for the different expression levels (low, medium, or high) of RasGRP1-EGFP. (A and D: WT N = 8, His 212 Tyr N = 8, His 212 Ala N = 6, His 212 Arg N = 3, His 212 Lys N = 3, B and E: WT N = 10, His 212 Tyr N = 7, His 212 Ala N = 7, C and F: WT, His 212 Arg, His 212 Lys, all N = 6). Kruskal-wallis test was used, with post-test Dunn's multiple comparisons, comparing each mutant to WT(*p<0.05), **p<0.01, ***p<0.001).