Figure 2. Rapid and titratable degradation of endogenous NCAPH and NCAPH2 in primary cells.

(A) Schematic illustration of experiments designed to test targeted degradation of condensin subunits in primary cells. (B) Western blots prepared from thymus whole cell extract and probed with polyclonal antibodies against NCAPH, NCAPH2, or a GAPDH loading control. Robust tag-dependent degradation of target proteins is clearly evident after 3 hr of auxin treatment. (C and D) Boxplots quantify the extent of targeted protein depletion following IAA treatment (500 μM for 3 hr), measured by flow cytometry in primary CD8⁺ thymocytes (C) and murine embryonic fibroblasts (MEFs - D). n = 3 biological replicates from at least 2 independent experiments, with degradation measured in over 1000 S/G2/M cells in each case. To calculate % protein remaining, the background-corrected fluorescence value of each cell was expressed as a percentage of the mean fluorescence value for all cells in the vehicle-only condition. Boxes show the boundaries of upper and lower quartiles and whiskers show the range. Where negative values were observed (e.g. in MEFs due to variable autofluorescence between lines), a value of 0% was assigned. (E) Titration of target protein levels in primary neural stem cells treated with different IAA concentrations for 2 hr. Boxplots were generated as described for panels C and D.