Stat5 Signaling Specifies Basal versus Stress Erythropoietic Responses through Distinct Binary and Graded Dynamic Modalities
Figure 4
Maximal p-Stat5 signal intensity (p-Stat5max) is linearly correlated with Stat5 protein levels.
(A) The p-Stat5 response of S1 cells from Stat5+/− fetal liver and from littermate wild-type controls. Representative p-Stat5 fluorescence histograms are shown for the indicated Epo concentrations. (B) Plots of “total p-Stat5 versus Epo concentration” in Stat5+/− and in wild-type littermate fetal livers in experiments similar to (A), fitted with Hill curves. Data (mean ± SE) from n = 7 Stat5+/− embryos and 6 littermate controls, each analyzed separately. p-Stat5 MFI was normalized as in Figure 1E. Parameter values used for fitting the Hill curves and goodness of fit information are in Figure S7A. (C) Plots of “total p-Stat5 versus Epo concentration” in EpoR+/− and in wild-type littermate fetal livers, fitted with Hill curves. Data (mean ± SE) from n = 4 EpoR+/− embryos and 3 littermate controls, each analyzed separately. p-Stat5 MFI was normalized as in Figure 1E. Parameter values used for fitting the Hill curves and goodness of fit information are in Figure S7C. (D) Stat5 protein levels in S1 to S3, in representative wild-type (WT) and Stat5+/− embryos, using flow-cytometric measurements with an anti-Stat5 antibody, characterized in Figure S7D using Stat5−/− embryos. FMO, “fluorescence minus one” control, in which cells were labeled for all parameters (Ter119, CD71, live/dead stain) except one: the anti-Stat5 antibody was omitted and replaced with an isotype-control antibody. (E) Stat5 protein levels in subsets S1 to S3 in wild-type or Stat5+/− embryos. Individual data points correspond to data from individual embryos, measured as in panel D. Stat5 protein is expressed as a ratio to the average fluorescence signal for S1 cells in all wild-type embryos. (F) Linear correlation between Stat5 protein levels and p-Stat5max, across all subsets in Stat5+/− and wild-type embryos (R2 = 0.85). Data points correspond to individual embryos. Stat5 protein levels are as in (D). p-Stat5max was determined by fitting Hill curves to individual embryo “total p-Stat5 versus Epo concentration” analyses, with p-Stat5 normalized as in Figure 1E.