Fluorescent Tags Can Impair DNA Mismatch Repair Figure 5. Putative three-dimensional structure models of fluorescent labeled MutLa. Using PyMol (Warren DeLano, http://www.pymol.org/), GFP or Red fluorescent proteins were attached to (A) N-termini of MLH1 and PMS2 or (B) C-termini of MutLa. C-terminal tags seem to hide the C-terminal region of MutLa and consequently might avoid DNA interaction. (C) Corresponding amino acid sequences of linker regions are shown. The dashed line between PMS2 and the N-terminal fluorescent tag illustrates a putative a-helix (unknown structure) of the first thirty amino acids of PMS2. doi:10.1371/journal.pone.0031863.g005 expression plasmids were described previously [10,22]. pECFP- N1- or pDSRed-N1-MLH1 (MLH1-GFP-C or MLH1-Red-C) and pECFP-N1- or pDSRed-N1-PMS2 (PMS2-GFP-C or PMS2- Red-C) were generated by subcloning of wild-type MLH1 [22] or PMS2 [22] using EcoRI and KpnI (DNA-cloning, Hamburg, Germany) into pECFP-N1 or pDSRed-N1 (Clontech Lab., Palo Alto, CA, USA). All used dye labeled MMR proteins are illustrated in Figure 1, confirmed by sequencing and reading frames were corrected using site-directed mutagenesis, if necessary. Oligonucleotides were purchased from Sigma-Aldrich (Munich, Germany). Antibodies, Western blot analysis, and protein quantification Anti-MLH1 (G168-728) was obtained from Pharmingen (BD Biosciences, United States), anti-PMS2 (E-19) was from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-b-Actin (Clone AC-15) was purchased from Sigma (Sigma-Aldrich, Munich, Germany). FITC-labeled goat anti-mouse IgG Alexa Fluor 488 as well as FITC-labeled goat anti-rabbit IgG Alexa Fluor 555 were obtained from Invitrogen (Darmstadt, Germany). Proteins were separated on 10% polyacrylamide gels, followed by Western blotting on nitrocellulose membranes and antibody detection using standard procedures. The band intensity of proteins was quantified using Multi Gauge V3.2 program (Fujifilm, Tokyo, Japan). All experiments were performed in quadruplicate. Confocal Laser Microscopy To analyze the influence of fluorescent labeling on cellular localization of MLH1 or PMS2 proteins, HEK293T cells were transiently transfected with unlabeled, GFP- or Red-labeled variants. Briefly, HEK293T cells were spread on coverslipes to 50% confluence 4 h prior transfection and transfected with expression plasmids (1 mg/ml, respectively) using 10 ml/ml of the cationic polymer polyethylenimine (Polysciences, Warrington, PA; stock solution 1 mg/ml). After 48 h, cells were prepared for confocal laser scanning microscopy essentially as described previously [10]. In brief, all cells transfected with fluorescent labeled as well as those transfected with unlabeled proteins were washed twice with PBS, fixed for 10 min in 3% formaldehyde in PBS, followed by permeabilization with 0.1% Triton X-100 for 5 min. Furthermore, cells transfected with unlabeled MLH1 or PMS2 were incubated for 1 h with the primary antibodies against MLH1 (1:1000) or PMS2 (1:1000) at room temperature, respectively. MLH1 antibody binding was detected with a FITC-labeled goat anti-rabbit IgG Alexa Fluor 488 (1:1000) whereas PMS2 antibody binding was detected with FITC labeled PLoS ONE | www.plosone.org 6 February 2012 | Volume 7 | Issue 2 | e31863