Fig. 5
From: Protein degradation kinetics measured by microinjection and live-cell fluorescence microscopy
Comparison of methods for degradation rate determination in living HEK293 cells using two representative DARPins (DP1 and DP2, see Supplementary Fig. S18d for sequences). Black lines in a to d show data fitting using an exponential decay model with (a, b) and without a free plateau parameter (c, d). Y-coordinates represent the relative amount of DARPin, normalized to the signal at timepoint zero. X-coordinates represent the time after the addition of cycloheximide (CHX) for western blot (WB, a) and luciferase assay (LUC, b). In microinjection assays using fluorescently labeled DARPin (with TMR, c) and DARPin genetically fused to mRuby3 (mR3, d), the x-axis indicates the time after the first recorded image after microinjection. For WB and LUC assays, data points and error bars shown in grey represent averages and standard deviations of replicates per timepoint. Pairwise statistical analysis for mR3 fusions and TMR-labeled constructs are shown in Supplementary Fig. S9. (f) For WB experiments, three biological replicates, each containing two technical replicates were performed. Bars represent the sum of pixel intensity after background subtraction, normalized to the first timepoint after ribosomal inhibition. Data are further adjusted to the total protein concentration of the samples, which were loaded in identical amounts. Original uncropped blots and regions of interest used for quantification are indicated and further discussed in Supplementary Fig. S24. (e) Extracted degradation rate constants obtained by all four methods are indicated as bars. (g) Fluorescence confocal micrograph for intracellular localization of fluorescently labeled DARPins, obtained six hours after microinjection.
