Figure 2.

Mechanism of Action of a JQ1-VHL PROTAC

(A) Schematic representation of the mechanism of action of a PROTAC.

(B) Scatterplots of protein fold changes (FC) observed in mature (upper panel) and nascent (lower panel) forms of proteins in THP-1 cells treated for 6 hr with the BET inhibitor JQ1-Az (10 μM), the JQ1-VHL PROTAC (1 and 10 μM), or VHL alkyne (10 μM) relative to vehicle control in two biological replicate experiments. Red closed circles indicate statistically significant regulation (p < 0.05), and BRD2, 3, and 4 and FYTTD1 are labeled exemplarily. The dashed diagonal indicates the equality line. Upper panels indicate regulation of proteins that were already present before compounds were added (mature proteins), and lower panels indicate proteins synthesized after compounds were added (nascent proteins). N indicates the number of proteins robustly quantified in both replicates.

(C) Line charts with markers of protein fold changes observed in mature (upper panel) and nascent (lower panel) forms of selected proteins after 6- (left) and 24-hr (right) treatment with JQ1-Az (10 μM, red), JQ1-VHL PROTAC (1 μM, blue; 10 μM, green), and VHL alkyne (10 μM, purple) relative to vehicle control. Triangles and circles distinguish data from biological replicates 1 and 2. Proteins where fold changes measured for both replicates are outside the dotted lines are significantly regulated (p < 0.05).

(D) Bar chart indicating relative contributions of nascent (green) and mature (orange) proteins to proteome composition after 6- or 24-hr incubation with vehicle, JQ1-Az (10 μM), and JQ1-VHL PROTAC (1 and 10 μM). Percentages of mature and nascent proteins are estimated from the summed-up reporter ion abundances.