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. 2022 Jun 23;11:e77987. doi: 10.7554/eLife.77987

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© 2022, Macdonald, Taylor et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 3. — (A) Schematic diagram illustrates the assembly of the Tir1 substrate receptor protein, IAA ligand and AID-tagged target protein-of-interest into a ternary complex necessary for target protein ubiquitination via SCF^Tir1, and degradation. (B and C) Histograms show the distribution of Clover expression levels, measured by flow cytometry in S/G2/M thymocytes cultured for 2 hr ex vivo in the presence of different IAA concentrations. Thymocytes were isolated from animals homozygous for either (B) Ncaph^AID:Clover or (C) Ncaph2^AID:Clover alleles in combination with either one (dark purple) or two (light purple) alleles of Rosa26^Tir1. Equivalent data from animals heterozygous for AID-tagged alleles are shown in Figure 5—figure supplement 1. (D) Comparison of depletion kinetics in the presence of one (black) versus two (red) alleles of the Tir1 transgene at low (solid line) versus high (dashed line) ligand concentrations (n = 3 biological replicate samples). Each experiment in panels B–D used data from at least 1000 S/G2/M thymocytes, gated on DNA content. In panel D, the mean background-corrected fluorescence value for each cell population is expressed as a percentage of the mean background-corrected fluorescence value for the vehicle only condition.

Figure 3—figure supplement 1. — (A) Schematic diagram illustrates the assembly of the Tir1 substrate receptor protein, IAA ligand and AID-tagged target protein-of-interest into a ternary complex necessary for target protein ubiquitination via SCF^Tir1, and degradation. (B and C) Histograms show the distribution of Clover expression levels, measured by flow cytometry in S/G2/M thymocytes cultured for 2 hr ex vivo in the presence of different IAA concentrations. Thymocytes were isolated from animals homozygous for either (B) Ncaph^AID:Clover or (C) Ncaph2^AID:Clover alleles in combination with either one (dark purple) or two (light purple) alleles of Rosa26^Tir1. Equivalent data from animals heterozygous for AID-tagged alleles are shown in Figure 5—figure supplement 1. (D) Comparison of depletion kinetics in the presence of one (black) versus two (red) alleles of the Tir1 transgene at low (solid line) versus high (dashed line) ligand concentrations (n = 3 biological replicate samples). Each experiment in panels B–D used data from at least 1000 S/G2/M thymocytes, gated on DNA content. In panel D, the mean background-corrected fluorescence value for each cell population is expressed as a percentage of the mean background-corrected fluorescence value for the vehicle only condition.