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. 2021 May 3;4:517. doi: 10.1038/s42003-021-02046-9

Fig. 6. Pleckstrin-2 interacts with and results in RacGTPase activation in erythroid precursors.

Fig. 6

a Western blot for Rac1 expression in bone marrow erythroblasts from WT, β-thalassemic, and apoTf-treated β-thalassemic mice. b Statistical analysis of (a) reveals no difference in Rac1 expression between groups (n = 3 mice/group; MW = Rac1 21 kDa, actin 42 kDa). c Confocal immunofluorescence microscopy of Rac1 and plek2 interaction (red) using the proximity ligation assay in sorted bone marrow Pro-Es from (2–3 mice pooled per data point, n = 3 data points per category) WT, β-thalassemic, and apoTf-treated β-thalassemic mice. Scale bar 5 µm. d Statistical analysis of (c) quantifying Rac1 and plek2 interaction, demonstrating increased interaction in Pro-E from β-thalassemic relative to WT mice, normalized after apoTf treatment. e FRET of activated Rac1 in erythroblasts from WT, β-thalassemic, and apoTf-treated β-thalassemic mice. Scale bar 10 µm. The images are representative cells from at least 20 cells analyzed in each condition. f Statistical analysis of (e) reveals more activated Rac1 in β-thalassemic relative to WT erythroblasts, normalized after apoTf treatment. All data are reported as mean ± s.e.m. and p < 0.05 is considered statistically significant (one-way ANOVA). g Activated Rac1 pull-down from fetal liver cells from E13.5 WT embryos transfected with nt-shRNA and plek2-shRNA (MW = plek2 38 kDa, Rac1 21 kDa, actin 42 kDa). h Statistical analysis of activated Rac1 FRET in WT and β-thalassemic erythroblasts (n = 4 mice/group) cultured for 24 h (early erythroblasts) reveals more activated Rac1 in β-thalassemic relative to WT. All data are reported as mean ± s.e.m. and p < 0.05 is considered statistically significant (Student’s unpaired t-test). All data are reported as mean ± s.e.m. and p < 0.05 was considered statistically significant (two-way ANOVA) (*p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005). WT = wild type; thal = β-thalassemic; apoTf = apo-transferrin; plek2 = pleckstrin-2; MFI = mean florescence index; Pro-E = pro-erythroblasts; Baso-E = basophilic erythroblasts; Poly-E = polychromatophilic erythroblasts; Ortho-E = orthochromatophilic erythroblasts; FLC = fetal liver cell.