Figure 4. Dosage of auxin-inducible degron (AID)-tagged substrate proteins determines degradation kinetics.
(A) The relative expression of NCAPH and NCAPH2 (n = 6 biological replicates each) in thymocytes, based on flow cytometric Clover fluorescence measurements in >1000 cells. (B) Table showing the relative total dose of AID-tagged proteins in mice heterozygous for Ncaph2AID:Clover in combination with either 0 (Low), 1 (Medium), or 2 (High) alleles of NcaphAID:Clover. Relative AID dose is calculated based on data in panel A. (C) Schematic showing the time course for auxin treatment of primary thymocytes in panels D and E. (D) Western blots probed with a polyclonal antibody against NCAPH2. Tagged protein (upper band) is degraded, whereas wildtype protein (lower band) is not. * indicates non-specific band. (E) Quantification of NCAPH2-AID:Clover depletion in the presence of low, medium, or high overall AID-tagged protein dose. Density of the AID:Clover band (see panel D) was first measured relative to the corresponding wildtype allele (bottom) as an internal control. The AID:WT ratio in the vehicle only control was set at 100% and IAA treatment conditions were then calculated relative to this value. Data from two independent experiments are presented.