The auxin-inducible degron (AID) system allows “on demand” depletion of the proteome. (A) Schematic representation of the AID system, where a protein of interest (POI) is fused to the AID2 tag and a GFP for visualization. In the same cells, the OsTIR1(F74G) adaptor protein is constitutively expressed. Upon induction by the addition of the modified auxin, 5-Ph-IAA, the construct gets ubiquitinated and degraded via the proteasome. (B) Fluorescent images of selected proteins tagged with the AID system after 0, 30, and 180 min of induction. All the proteins are depleted either partially or below detection levels upon induction. For Gpp1 and Npl3 at 180 min, the outline of the cells is depicted with a dotted line. Scale bar: 5 µm. (C) Western blots (WB) of the same strains shown in panel B. Immunoblotting was performed against GFP (green) and actin (magenta) as a loading control. The GFP-tagged proteins are depleted partially or below detection levels upon induction. Only one band is detected, at the expected molecular weight in each case, indicating no free GFP or intermediate degradation stages are generated. (D) Graphs comparing the degradation curves for each protein from panel B, based either on the fluorescence microscopy images (yellow circles) or on the western blots from panel C (pink squares), normalized to time 0. Both methods produced comparable curves, despite the half-life (t_50M for microscopy and t_50W for WB) of the proteins spanning a wide range of time. Three technical replicates were used in each case. A one-phase decay curve was fitted to the data to estimate the half-lives of each protein and the 95% confidence interval of the t₅₀ is displayed. Source data are available for this figure: SourceData F1.