Fig. 1: eGFP tagging of RAG1 and RAG2 sheds light on expression and nuclear localization.

a RAG1 or RAG2 variants were tagged with eGFP on the C-terminus and integrated in BY4742-NAB2-mCherry yeast. Median eGFP fluorescence was then measured using flow cytometry after overnight culture in YPG. b Confocal microscopy images of eGFP-tagged RAG1 and RAG2 protein variants (green) in a NAB2-mCherry strain which natively highlights the nucleus (red). Scale bar represents 5 μm; all images are at the same scale. Gains have been adjusted from the original images to aid visual comparison. The brightfield channel for each image can be found in Supplementary Fig. S1. c Nuclear localization was estimated by calculating the Pearson correlation coefficient between the RAG-eGFP and NAB2-mCherry signals of the confocal images. d Confocal microscopy images of eGFP-tagged RAG1 protein variants (green) in a NOP56-mCherry strain which natively highlights the nucleolus (red). As in (b), scale bar represents 5 μm. The complete set of images can be found in Supplementary Fig. S2. e Nucleolar localization was estimated by calculating the Pearson correlation coefficient between the RAG-eGFP and NOP56-mCherry signals of the confocal images. RFU = relative fluorescence units, NLS = nuclear localization signal. In (a, c, and e) data are presented as mean values ± SD; n = 3 biological replicates. Statistical significance was calculated with a one-way ANOVA and Tukey test (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001). From left to right, the highlighted p values are, in (a), p = 0.0014, <0.0001, 0.0012, and 0.0001; in (c), p = <0.0001, 0.0499, 0.0016, and 0.0194; and in (e), p = <0.0001 and <0.0001. Source data are provided in the Source Data file.