Fluorescent Tags Can Impair DNA Mismatch Repair Figure 2. Influence of fluorescent labeling of MutLa on protein expression levels. To analyze the effect of fluorescent dyes on protein expression levels, HEK293T cells were transiently cotransfected with different MutLa constructs (see below) and (A) Western blot analysis was carried out after 48 h using anti-MLH1 or anti-PMS2, respectively, controlled by b-actin detection. (B) Amounts of expressed proteins were assessed by measuring the signal intensities of protein bands with Multi Gauge V3.2 software. Graphs indicate the results (mean 6S.D.) of at least four independent experiments in which the proportion of protein expression using an unbiased method were presented. 1: negative control (untransfected). 2: MLH1/PMS2 unlabeled; 3: MLH1/PMS2-GFP-N; 4: MLH1/PMS2-GFP-C; 5: MLH1/PMS2-Red-N; 6: MLH1/PMS2-Red-C; 7: MLH1-GFP-N/ PMS2; 8: MLH1-GFP-C/PMS2; 9: MLH1-Red-N/PMS2; 10: MLH1-Red-C/PMS2; 11: MLH1-GFP-N/PMS2-Red-N; 12: MLH1-GFP-N/PMS2-Red-C; 13: MLH1- GFP-C/PMS2-Red-N; 14: MLH1-GFP-C/PMS2-Red-C; 15: MLH1-Red-N/PMS2-GFP-N; 16: MLH1-Red-N/PMS2-GFP-C; 17: MLH1-Red-C/PMS2-GFP-N; 18: MLH1-Red-C/PMS2-GFP-C. Symbols see Figure 1. doi:10.1371/journal.pone.0031863.g002 PLoS ONE | www.plosone.org 3 February 2012 | Volume 7 | Issue 2 | e31863