Extended Data Fig. 8: CCL22-CCR4 downstream signaling.
a, Phosphorylation of AKT, and mitogen-activated protein kinase (MAPK) (p44/42 extracellular signal-regulated kinase (Erk) 1/2) by immunoblotting. Ba/F3-CCR4 cells were stimulated with exogenous recombinant wild type or mutant CCL22 (50 ng/ml) for 5 minutes (min). Both exogenous recombinant wild type and mutant CCL22 activated pAKT in a similar way, but p42/44 ERK was slightly reduced by mutant CCL22. The samples derive from the same experiment and were processed in parallel. b, Ba/F3-CCR4 cells were stimulated over time with exogenous recombinant wild type or mutant CCL22 (100 ng/ml) and pAKT was assessed by phosphoflow cytometric analysis. Activation of pAKT was not different between exogenous recombinant wild type and mutant CCL22. The mean (± SD) fluorescence intensity (MFI) is shown (n = 3). c,d, Single cell phosphoproteomic detection via IsoLight validated reduced phosphorylation of ERK in mutant CCL22 (50 ng/ml) after three-hour (h) incubation. c, Positive cell rate of pERK and (d) the mean of detected pERK signal are shown. P value was calculated by t-test. **0.001 < p < 0.01.
