No induction of expression of myelomonocyte-specific and B-cell–specific genes in MEL cells after overexpression of a mutant PU.1 with part of the activation domain deleted (PU.1-ΔA).
(A) Total RNA was extracted from PU.1-ΔA-1 cells cultured for 48 hours without reagents (lane 1), with DMSO (lane 2), with ZnCl2 (lane 3), or with DMSO and ZnCl2 (lane 4). Expression of myelomonocyte-specific and B-cell–specific genes was examined by Northern blot analysis. Total RNA from PU.1 cells cultured for 48 hours with ZnCl2 was used as the positive control (lane 5). (B) Total RNA was extracted from PU.1-ΔA-1 cells cultured for 48 hours without ZnCl2 (lane 1) or with ZnCl2 (lane 2). Expression of myelomonocyte-specific and B-cell–specific genes was examined by RT-PCR analysis. Although the photograph is separated between lanes 1 and 2, PCR products of each gene were run in the same gel. (C) Total RNA was extracted from PU.1-ΔA-2 cells cultured for 48 hours without ZnCl2 (lane 1) or with ZnCl2 (lane 2). Expression of myelomonocyte-specific and B-cell–specific genes was examined by RT-PCR analysis. (D) Total protein was extracted from PU.1-1 cells (lanes 1 and 2), PU.1-ΔA-1 cells (lanes 3 and 4), and PU.1-ΔA-2 cells (lanes 5 and 6) cultured for 8 hours without reagents (lanes 1, 3, and 5) or with ZnCl2 (lanes 2, 4, and 6). Expression of wild-type and mutant PU.1 proteins was examined by Western blot analysis.
