The G208fsX mutant is defective in induction of myeloid differentiation of PU.1^−/− cells.

Conditional expression of the estrogen receptor alone, or fused to wild-type PU.1 or the G208fsX mutant in PU.1^−/− cells. (A) Left panel: Western blot using carboxyl terminal PU.1 antiserum (1:500; Santa Cruz, catalog #sc-352) of whole cell lysates from PU.1^−/− cells transfected with wild-type human PU.1-estrogen receptor fusion plasmid (wt-ER) or the estrogen receptor (V-ER) alone. The V-ER estrogen receptor alone contains no PU.1 or FLAG sequences. The migration of molecular weight markers is shown to the left of each panel. The blot was reprobed for β-tubulin as a loading control (lower panel). Right panel: Western blot using a FLAG antibody detecting the G208fsX PU.1 mutant fused to the estrogen receptor. Shown below is the β-tubulin control. (B) Flow cytometric analysis for CD11b expression (upper panel) and G-CSF receptor expression (lower panel). PU.1^−/− cells expressing the PU.1 wt-ER, the PU.1 mutant G208fsX-ER, or the estrogen receptor alone (V-ER) were untreated (fine lines) or treated (thick lines) with 1 mM β-estradiol for 7 days. CD11b and G-CSF receptor expression were determined by flow cytometry. (C) Wright-Giemsa staining of PU.1^−/−cells expressing PU.1 wild-type or mutant-ER fusion proteins. Cells were untreated (d7-EST) or treated with (d7 + EST) 1 mM β-estradiol for 7 days. The arrow in the upper right panel indicates a mature neutrophil in cells expressing PU.1 wild-type protein. Magnification × 100.