Figure 3.

Stable antigen is the major source of MHC class I presentation upon degradation tag (dTAG)-7 treatment. (A) Real-time PCR analysis to measure the stability of GFP-S8L-F12 mRNA. BMC-2 cells were treated with 1 µg/mL doxycycline to induce GFP-S8L-F12 expression for 24 h. Then, doxycycline was washed off and mRNA stability was followed for indicated times. GFP-S8L-F12 expression is shown as fold induction compared to basal levels in the absence of doxycycline (- Dox). (B) Schematic illustration of the experiments performed in (C,D). (C,D) GFP-S8L-F12 expression in BMC-2 (C) and MC57G cells (D) was induced by doxycycline for 48 h. Next, doxycycline was removed for 4 h, before cells were treated with 1 µM dTAG-7 for 3 or 6 h, respectively. Afterward, GFP-specific fluorescence (MFI-GFP) and MHC class I presentation of S8L (MFI-H-2K^b/S8L) were quantified by flow cytometry. MFI, mean fluorescence intensity. (E) Quantification of the results shown in (C,D) from three independent experiments (for calculation details see Materials and Methods). All experiments were performed in triplicates and results are shown as mean ± SEM. Representative results from three independent experiments are shown.