NBN variant protein stability screen. (A) Presentation of the fold change of NBN variant EGFP:mCherry ratio compared with WT NBN as quantified by flow cytometry: complete loss <0.1, partial loss 0.1 to 0.5, mild loss 0.5 to 0.8, WT-like >0.8. The results are presented as the mean of 3 independent experiments ± standard deviation. (B) The pattern of EGFP:mCherry distribution in unstable NBN variants (red) compared with WT NBN (green). Each histogram was generated from ∼4000 NBN (WT or variant) expressing cells. (C) Western blot analysis of NBN (WT or variant) protein expression level. Lanes 1 to 2 show the absence of WT NBN protein expression in NBN^−/− HEK293T LP cells (lane 2) compared with WT HEK293T LP cells (lane 1). Lanes 3 to 5 depict NBN (WT or variant) protein levels after reexpression in NBN^−/− HEK293T LP cells. Successfully recombined cells were identified as mCherry⁺/BFP⁻ population and separated by flow cytometry before protein extraction and western blot analysis. The EGFP-fusion protein resulted in a slight increase in the molecular weight. (D) Relative NBN variant expression level compared with WT NBN expression as quantified using western blot.