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. 2004 Jan;24(2):675–686. doi: 10.1128/MCB.24.2.675-686.2004

FIG. 6.

FIG. 6.

C/EBPα-S21D is unable to induce granulopoiesis in K562 cells. (A) K562 cells were stably transfected with C/EBPα-ER fusion proteins encoding either wild-type (WT) C/EBPα or a C/EBPα mutant form in which serine 21 was replaced with alanine (S21A) or aspartate (S21D). Lysates from the parental K562 line (−) and two independent stable lines for each C/EBPα-ER fusion protein (wild-type and S21Aand S21D mutant forms, as indicated) were separated by SDS-PAGE and subjected to immunoblot analysis with an antibody against C/EBPα. (B) C/EBPα-ER-expressing stable lines from panel A were left untreated (control [Con]) or treated with 1 μM β-estradiol (Est) for 24 h. Cells were fixed and visualized by immunofluorescence with an antibody to C/EBPα. (C) C/EBPα-ER-expressing stable lines from panel A were left untreated (Con) or treated with 1 μM β-estradiol (Est) for 3 days, at which point cells were analyzed for NBT reduction. (D) Cells were scored for reduction of NBT, and quantified data are presented. (E) C/EBPα-ER-expressing stable lines from panel A were left untreated (−) or treated with 1 μM β-estradiol (+) for 3 days. RNA was collected and analyzed by Northern blotting with probes for the G-CSF receptor (GCSFR), C/EBPɛ, and GAPDH.