Figure 1.
Testing functionality and expression of GFP-tagged MN1, TEL and MN1-TEL.
A. GFP-MN1 stimulates transcription from the MSV promoter as efficiently as MN1, both in the presence or absence of 1 uM ATRA (24 h incubation) as tested in with a luciferase assay. Western blot using anti-GFP antibody showing GFP-MN1 protein expression in stably transfected NIH 3T3 cells. GFP-MN1 located to the nucleus, where it was homogeneously distributed, with notable exception of the nucleoli, where no expression could be detected (left photograph). This expression pattern did not change upon alpha-amanitin treatment (right photograph). B. TEL and TEL-GFP are equally able to inhibit gene expression from the MSV promoter. Western blot of a cell lysate of the TEL-GFP-expressing NIH3T3 cell line is stained with anti-GFP and the two isoforms (translation starts at methionine 1 and 43) of TEL are visible. TEL-GFP is mostly nuclear in NIH3T3 cell lines (left photograph). TEL-GFP-DBDm is located in the cytoplasm (middle photograph). Expression pattern of TEL-GFP upon treatment with 50 ug/ml alpha-amanitin for 3 h (right photograph). Both MN1-TEL and GFP-MN1-TEL can weakly stimulate transcription from the MSV promoter, while the DNA-binding mutant is a weak repressor. Western blot with anti-GFP antibody of a lysate of cells expressing GFP-MN1-TEL and GFP-MN1-TEL-DBDm. GFP-MN1-TEL is nuclear, with a tendency to form aggregates upon higher expression levels (upper two photographs). GFP-MN1-TEL-DBDm is both nuclear and cytoplasmic (left lower photograph). Expression pattern of GFP-MN1-TEL did not change upon treatment with 50 ug/ml alpha-amanitin for 3 h (right lower photograph).
Figure 2.
FRAP curves and simulation data.
A. GFP-MN1 FRAP curves show the fast recovery of MN1 to levels similar as GFP. In the presence of ATRA the recovery of GFP-MN1 is slightly slower. Alpha-amanitin releases the MN1 protein. Pie diagrams containing simulation data support FRAP curve data. B. TEL-GFP recovered much slower and leveled off to 70% of pre-bleach values. Simulation data calculated the presence of both a long-term bound fraction (20%; 59 s) and a short-term bound fraction (57%; 0.63 s). Alpha amanitin released the TEL-GFP protein as shown by FRAP curve and simulation data. C. GFP-MN1-TEL was largely immobile. A non DNA-binding mutant (DBDm) of GFP-MN1-TEL was mobile and diffused similarly to GFP-MN1. Alpha-amanitin treatment only partly released MN1-TEL. Simulation data calculated that 10% of the protein remains immobile for a long period (235 s).
Figure 3.
Stromelysin promoter is inhibited by TEL and stimulated by MN1-TEL.
Expression and reporter plasmids, 100 ng and 250 ng respectively, were transiently transfected in NIH-3T3 cells. Expression of TEL results in inhibition of stomelysin promoter, whereas MN1-TEL stimulates. The non-DNA-binding mutant of MN1-TEL failed to stimulate. Equal amounts of TEL and MN1-TEL show intermediate promoter activity, indicative of competition of the proteins for the same binding sites.