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. Author manuscript; available in PMC: 2013 Feb 3.
Published in final edited form as: Stem Cells. 2009 Dec;27(12):2884–2895. doi: 10.1002/stem.203

Figure 3. Mixl1 functions as a transcriptional activator in cultured NIH 3T3 cells.

Figure 3

(A) Diagram of the plasmids for transient transfection of NIH 3T3 cells. The promoterless pGL3-basic served as a negative control. pGL2pro contains a modified SV40 promoter (black box with arrow) driving the firefly luciferase gene as the reporter. pGL2pro-6xMBS contains six tandem copies of the MBS (6xMBS) upstream of the SV40 promoter and the reporter gene. (B) NIH 3T3 cells were co-transfected by pGL3-basic, pGL2pro or pGL2pro-6xMBS with either pMT23 or pMT23-FLAG-Mixl1, that encodes a FLAG-tagged Mixl1 protein. Mixl1 induced modest activation of the SV40 promoter alone (pGL2pro) but strong activation of the promoter-reporter gene linked to the multimerized MBS (pGL2pro-6xMBS). (C) Sequence of the Mixl1 homeodomain (residues 86–145) is shown. Either the P126I or V132A mutation abolished the DNA binding activity of Mixl1. (D) NIH 3T3 cells were transfected with pMT23 (vector), pMT23-FLAG-Mixl1 (Mixl1), pMT23-FLAG-Mixl1 P126I (P126I), or pMT23-FLAG-Mixl1 V132A (V132A), and the expression of FLAG-tagged Mixl1 and its mutant forms were detected by Western blotting using anti-Mixl1 antibody. The FLAG-Mixl1 (lane 2), FLAG-Mixl1 P126I (lane 3), and FLAG-Mixl1 V132A (lane 4) polypeptides are marked by an arrow. Endogenous β-Actin (loading control) was detected using an anti-β-Actin antibody. (E) NIH 3T3 cells were co-transfected with the reporter plasmid (pGL2pro or pGL2pro-6xMBS) and pMT23, pMT23-FLAG-Mixl1 (Mixl1), pMT23-FLAG-Mixl1 P126I (Mixl1 P126I), or pMT23-FLAG-Mixl1 V132A (Mixl1 V132A). Wild type Mixl1 but not either of the Mixl1 mutants (Mixl1 P126I and Mixl1 V132A) activated the reporter gene (pGL2pro-6xMBS).