Figure 6. Rapid degradation of endogenous tagged proteins in living mice.

(A) (Top) I.P. injection time course to test protein degradation in vivo. Each mouse received a single injection of IAA solution (100 mg/kg), or vehicle. (Bottom) Boxplots show the extent of targeted protein degradation in >1000 S/G2/M CD8⁺ thymocytes harvested 1 or 2 hr following auxin injection, measured by flow cytometry. % protein remaining was calculated as described in Figure 2 legend. Boxes indicate the boundaries of upper and lower quartiles and whiskers show the range. Data are from three biological replicate injections performed over at least two independent experiments. (B) Proteome quantification by mass spectrometry analysis of MACS-purified CD8⁺ thymocytes. n = 3 animals per condition. (C) Protein degradation and recovery following a single I.P. injection. Data are presented as described for panel A, except mice were heterozygous for the Ncaph2^AID:Clover allele. (D) Schematic illustration of experimental workflow for protein degradation in E10.5 embryos. (E) Example image from whole-mount immunofluorescence performed on E10.5 embryo cryosections, stained with DAPI, anti-GFP-647 nanobooster (detecting NCAPH-AID:Clover), and anti-CDH1. Anti-GFP signal was quantified within five CDH1⁺ regions of interest (ROI) per embryo, which were selected based solely on the CDH1 staining pattern. To enable CDH1 localisation and ROIs to be visualised, the anti-GFP-647 channel is not shown in this panel. Images were captured at ×40 magnification, scale bar = 800 μm. (F) Example ROI’s from CDH1⁺ stained tissue on which target protein quantification was performed. To visualise degradation, only the NCAPH-AID:Clover channel is shown. Scale bar = 10 μm. (G) Quantification of degradation efficiency in CDH1⁺ embryonic cells. Mean pixel intensity was first calculated from five Cdh1⁺ regions in Ncaph^{AID:Clover/AID:Clover} Rosa26^Tir1/Tir1 embryos from mothers injected with either IAA or vehicle, and non-fluorescent negative control embryos (n = 1 embryo each). The mean pixel intensity value from negative control ROIs was set to 0%, and the mean value from vehicle-only ROIs to 100%. Mean pixel intensity values for each ROI from vehicle and IAA-exposed embryos were then plotted on this scale. Negative values were set to 0%.

Figure 6—figure supplement 1. Rapid degradation of endogenous tagged proteins in living mice.

(A) Boxplots quantify the extent of targeted protein depletion in CD8⁺ thymocytes from Ncaph^AID:Clover/+Rosa26^Tir1 animals injected with IAA at increasing dose. % protein remaining was calculated as described in the legend for Figure 2. Boxes show the boundaries of upper and lower quartiles and whiskers show the range. Where negative values were observed, a value of 0% was assigned. N = 1 per condition. (B) A panel of liver function tests performed on plasma collected post-mortem from adult mice (n = 3 per condition) 2 or 72 hr after I.P. injection with IAA (100 mg/kg) or vehicle. No significant differences (p < 0.05) were detected in unpaired two-tailed t-tests. ALP: alkaline phosphatase; AST: aspartate transaminase; ALT: alanine transaminase. (C) I.P. injection time course to test protein degradation in CD19⁺ bone marrow cells in vivo. Data were captured, analysed and presented as described in Figure 6A.(D) Schematic illustration of experimental workflow for protein degradation in E10.5 embryos. (E) Example image from whole mount immunofluorescence performed on E10.5 embryo cryosections, stained with DAPI, anti-GFP-647 nanobooster (detecting NCAPH-AID:Clover) and anti-PDGFR. Anti-GFP signal was quantified within 5 PDGFR⁺ regions of interest (ROI) per embryo, which were selected based solely on the PDGFR staining pattern. To enable PDGFR localisation and ROIs to be visualised, the anti-GFP-647 channel is not shown in this panel. Images were captured at 40X magnification, scale bar = 800μm (F) Example ROI’s from PDGFR⁺ stained tissue on which target protein quantification was performed. To visualise degradation, only the NCAPH-AID:Clover channel is shown. Scale bar = 10μm (G) Quantification of degradation efficiency in PDGFR⁺ embryonic cells. Mean pixel intensity was first calculated from 5 PDGFR⁺ regions in Ncaph^{AID:Clover/AID:Clover} Rosa26^Tir1/Tir1 embryos from mothers injected with either IAA or vehicle, and non-fluorescent negative control embryos (n = 1 embryo each). The mean pixel intensity value from negative control ROIs was set to 0%, and the mean value from vehicle-only ROIs to 100%. Mean pixel intensity values for each ROI from vehicle and IAA-exposed embryos were then plotted on this scale.

Figure 6—figure supplement 2. Rapid degradation of endogenous tagged proteins in living mice.

(A) Immunofluorescence on cryosections from small intestine of an Ncaph^AID/AID Rosa26^TIr1/Tir1 adult, fixed following 2-hr exposure to IAA in vivo (100 mg/kg, I.P.). Scale bar 10 μm. NCAPH degradation was quantified specifically within DAPI-stained regions of mitotic (ph3S10⁺) cells, but can also be observed in the vast majority of interphase cells. Arrows show the position of an IAA-unresponsive cell. (B) Different levels of NCAPH degradation observed in CD8⁺ thymocytes and Ter119⁺ erythroblasts from a single animal 2 hr following I.P. injection of IAA. (C) Western blots probed with anti-myc (top) to detect Tir1 expression, and anti-pan histone H3 loading control (bottom) in whole cell extracts from MACS-purified blood cell populations shown in panels B and D. (D) Different levels of NCAPH degradation in CD19⁺ B-cell precursors and Ter119⁺ erythroblasts following IAA treatment from the same ex vivo short-term bone marrow culture. In panels B and D, boxes show the boundaries of upper and lower quartiles and whiskers show the range of degradation values for >1000 S/G2/M cells, calculated as described in the legend for Figure 2. (E) Immunofluorescence on cryosections from fixed adult testes (Ncaph^AID/AID Rosa26^TIr1/Tir1) shows little if any target protein degradation. Yellow boxes in the upper panel show zoomed regions in the lower panel. Upper scale bar = 15 μm, lower scale bar = 10 μm.

Figure 6—figure supplement 3. General schematic and timeline for generating germline transgenic mice for studying protein function using the auxin-inducible degron (AID) system.

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