c-Maf in myeloid cell lines and stable cell clones. (A) Endogenous expression of c-Maf mRNA expression levels in human myeloid cell lines as determined by Northern blot analysis of polyA⁺ RNA from the HL-60 promyelocytic or U937 monoblastic cell lines. A single blot was initially probed with the c-Maf specific cDNA probe, stripped, and reprobed with c-Myb, and finally, β-actin as a control for RNA loading and integrity. Exposure times for optimal detection of c-Maf mRNA were routinely significantly longer than for other probes (1 week v 1 day). (B) Conditional expression c-Maf in stably transfected clones. Individual HL-60 or U937 clonal cell lines containing either CB6 vector alone (left 2 lanes) or c-Maf (right lanes) encoding plasmids were cultured for 24 hours with 150 μmol/L ZnSO₄. Cell lysates were analyzed beside control lysates from a previously established c-Maf inducible cell line (+).9 The gels were transferred and probed for c-Maf expression with an antiserum that recognized the c-Maf protein. (C) Myb:Maf complexes increase on c-Maf induction. An end-labeled 129-bp promoter fragment probe containing the functionally definedCD13/APN myeloid promoter elements9 was incubated with uninduced (-) or induced (+) whole-cell lysates from control vector-containing (CB6, lanes 2 and 3), c-Maf–containing (c-Maf clone 6, lanes 4 to 8) stable HL-60 clones. Unlabeled competitor oligonucleotides containing either the consensus Myb site from themim-1 promoter (mimAmyb, lane 6) or the consensus Maf binding site (MARE, lane 8) were added to the assays in 100-fold molar excess before addition of probe. Maf binding to its consensus oligonucleotide interferes with its ability to complex with Myb.9Antibodies recognizing the c-Maf protein (lane 7) were added to binding reactions before probe addition. (D) The increase in Myb:Maf complexes is dose-dependent and comparable to induction on monocytic differentiation. The identical promoter fragment probe used in (C) was incubated with uninduced (0) or induced (indicated concentrations) whole-cell lysates from control vector-containing (CB6, lanes 10 and 11), c-Maf–containing (c-Maf, lanes 12 to 16) stable HL-60 clonal lines or from untreated (lane 17), TPA-treated (lane 18) HL-60 parental cells.