Figure 5.

A fluorescence-based system to monitor FAAP20 stability. A, schematic of the FAAP20–P2A–EGFP reporter construct. A CMV promoter drives the transcription of the single mRNA transcript for both Flag–FAAP20 and EGFP. Ribosomal skipping of glycyl-prolyl peptide bond formation in a P2A “self-cleaving” peptide leads to the generation of Flag–FAAP20 and EGFP polypeptides at an equal molar ratio. EGFP serves as an internal control for transfection, whereas Flag–FAAP20 is stained by a PE-conjugated anti-Flag antibody for fluorescence quantification. B, left panel: a representative FACS plot showing Flag–PE⁺/EGFP⁺ cells in the upper right quadrant. Right panel: immunoblots confirming the production of Flag–FAAP20 and EGFP in U2OS cells transfected with Flag–FAAP20–P2A–EGFP. C, left panel: FACS histogram of Flag–PE and EGFP from U2OS cells transfected with the indicated amount of the Flag–FAAP20–P2A–EGFP reporter construct. Ctrl-PE indicates cells stained with a PE-conjugated IgG2a control antibody. Right panel: mean PE/EGFP values from the various amounts of plasmid transfection are plotted. D, left panel: U2OS cells expressing the reporter construct were treated with 10 μm MG132 for 6 h and analyzed by FACS. Right panel: fold-change of the Flag–PE mean value normalized by EGFP. Error bar indicates mean ± S.D., n = 2 from two independent experiments, **, p < 0.01, Student's t test. E, left panel: U2OS cells expressing the reporter construct were treated with 100 μg/ml of CHX for 5 h and analyzed by FACS. Right panel: fold-change of the Flag–PE mean value normalized by EGFP. Error bar indicates mean ± S.D., n = 3 from three independent experiments, **, p < 0.01, Student's t test.