Fig. 2: Regulation of eDHFR-YFP in JURKAT and HEK293T cells with TMP PROTACs.

A Schematic of eDHFR-YFP-T2A-Luciferase construct. YFP is directly fused to the C-terminus of eDHFR to allow for regulation of the fluorescent protein with TMP PROTACs. B Screening of compounds 7a-c and e in JURKAT-eDHFR-YFP cells. JURKAT-eDHFR-YFP cells were incubated with TMP-PROTACs for 4, 8, and 24 h. YFP fluorescence assessed by flow cytometry showed effective degradation of eDHFR-tagged YFP by 7b and 7c. Representative data from n = 3. C Dose-response screening of compounds 7a, 7b, and 7e at 24 h post-incubation. Degradation of eDHFR-tagged YFP in HEK293T cells analyzed by Western blot with anti-GFP antibody. eDHFR-YFP protein fusion is 45 kDa. COX IV = loading control, 18 kDa. n = 3. D Dose–response of 7c in HEK293T-eDHFR-YFP cells showed robust dose-dependent degradation of eDHFR-tagged YFP at 24 h post-incubation. eDHFR-YFP detected with anti-GFP antibody. eDHFR-YFP protein fusion is 45 kDa. COX IV = loading control, 18 kDa. n = 3. E Dose–response and time course of 7c in JURKAT-eDHFR-YFP. Kinetics of YFP degradation by the lead compound 7c was characterized by incubating JURKAT-eDHFR-YFP cells with various doses of 7c for 4, 8, 12, 24, and 48 h and assessing for YFP expression with flow cytometry. Representative data from n = 3. F Dose–response and time course of 7c in HEK293T-eDHFR-YFP cells at 6, 12, and 24 h post-incubation. eDHFR-YFP detected with anti-GFP antibody. eDHFR-YFP protein fusion is 45 kDa. COX IV = loading control, 18 kDa. n = 3. G Reversal kinetics of YFP degradation in JURKAT-eDHFR-YFP cells. JURKAT-eDHFR-YFP cells were incubated with 100 nM of 7c and the YFP expression was monitored at several time points before and after drug washout. Representative data from n = 3. H Western blot analysis of YFP recovery in HEK293T-eDHFR-YFP cells incubated with 100 nM of 7c. eDHFR-YFP detected with anti-GFP antibody. eDHFR-YFP protein fusion is 45 kDa. COX IV = loading control, 18 kDa. n = 2.