Figure 4. Assessment of PROTAC-mediated Nef protein loss.

A) Flow cytometry of Nef-eGFP protein loss. CEM/Nef-eGFP cells were treated with doxycycline to induce expression of Nef-eGFP under conditions that result in a moderate level of positive cells by flow cytometry (see Figure 3A). Triplicate cultures of cells were treated with the Nef PROTAC analogs indicated at a final concentration of 3 μM, and 24 h later the percent of cells showing loss of Nef-eGFP protein expression were calculated relative to the DMSO controls and are presented as the mean value ± SE; individual data points are also shown. B) Correlation analysis of cell-surface CD4 rescue vs. Nef-eGFP protein loss (red data points, left) and MHC-I rescue vs. Nef-eGFP protein loss (blue data points, right). CD4 rescue was best-fit by linear regression, while MHC-I rescue showed a plateau effect. C) Immunoblot analysis. Cells expressing Nef-eGFP were treated as in part A with the eight active PROTACs, and lysates were prepared 48 h later for immunoblot analysis with Nef and Actin antibodies. A representative blot is shown. D) Immunoblot analysis was performed in duplicate, and band intensities were quantified by LI-COR infrared imaging and used to calculate Nef to Actin protein expression ratios. The bar graph shows the mean value for each ratio along with the individual values. The structures of the analogs with little to no activity in this assay (FC-13890, FC-14373, FC-14379, FC-14388) are shown in the Supplemental Information, Figure S2.