Molecular Characterization of the Schistosoma mansoni Zinc Finger Protein SmZF1 as a Transcription Factor
Figure 2
SmZF1 has a nuclear localization in COS-7 cells.
(A) COS-7 cells were transfected either with pEYFP-c1 (control) or pEYFP-SmZF1 plasmids for transient expression of the proteins YFP or YFP-SmZF1. After 48 h the cells were fixed with paraformaldehyde, quenched, permeabilized and the cells nuclei stained using Hoechst 33342 dye. Images were directly captured at 200x magnification using Carl Zeiss LSM 510 META. In order to obtain a clearer visualization of fluorescent signals in merged images, the original yellow fluorescence of YFP and blue fluorescence of Hoechst were modified to green and red, respectively, using Adobe Photoshop CS2. (B) SmZF1-transfected and control COS-7 cells were used to generate the total (TE), cytoplasmic (CE) and nuclear (NE) extracts. The extracts were submitted to 10% SDS-PAGE and blotted onto nitrocellulose membranes. Membranes were blocked overnight and samples were reacted with anti-myc, anti-GFP or anti-c-erbB-2 peroxidase conjugated antibodies (1∶1000). Subsequently, blots were washed and developed with ECL enhanced chemiluminescence reagents and exposed to X-ray films. The exclusively cytoplasmic human oncoprotein c-erbB-2 was used as a quality control for extracts. YFP can be visualized as an approximately 30 kDa protein and the recombinant protein YFP-SmZF1 as an approximately 50 kDa protein.
