Fig. 4: Assessment of protein expression and function.

a Expression of endogenous GATA2 protein in platelets of P9 (c.351C>G, p.T117T). Left: Representative western blot image for the expression level of GATA2 and ß-actin. Right: Relative optical density of GATA2 protein normalized with ß-actin. GATA2 deficiency group comprises four pathogenic mutations in GATA2 coding region. b Transactivation activity measured by GATA reporter assay. Previously reported p.L359V mutant was used as a positive control. Fold activation of luciferase reporter was determined from the triplicate values from three independent experiments and presented as the mean ± SD values. Comparison between the GATA2 WT and each synonymous Mut, as well as p value calculation was performed using a standard one-way ANOVA test. c The electrophoretic mobility shift assay (EMSA) depicting DNA-binding ability of GATA2 p.L217L, compared with WT and a known loss-of-function mutation p.R396Q, lanes spliced from the same gel runs (experiment repeated three times). Shift: semiquantitative comparison of GATA2 binding strength to the target oligo (biotin-labeled probe containing wild-type target sequence); Super Shift: specificity of FLAG-GATA2 binding to DNA sequence with anti-FLAG antibody (Ab); Competition: includes 10× and 100× excess of unlabeled wild-type probe competing for binding of the protein; Specificity: contains biotin-labeled probe with mutated target sequence which cannot be bound by the analyzed protein. Three latter setups verify that the signal obtained in the shift reaction is the result of specific DNA-protein interaction. d Assessment of translation efficiency (left) and protein stability (right) of GATA2 synonymous mutants in transiently transfected 293T cells treated with 1 µg/ml actinomycin D and 10 µg/ml cycloheximide, respectively. Experiments were performed in duplicates. Blots from representative experiments are shown.