Meso scale discovery (MSD) assays

All incubations were done on a Bellco Biotechnology orbital shaker at 1600 revolutions per minute. Washes were done with 200 µL of PBS-T (phosphate-buffered saline [pH = 7.4] with 0.05% tween-20). Plate coating was done in PBS. Blocking was done with 150 µL Blocker Casein in TBS (ThermoFisher) for 1 h at room temperature or overnight at 4°C. All antibody incubations were done in blocking buffer. Cells (30,000 cells per well in a final volume of 100 µL) were plated in 96-well tissue culture plates that had previously received 10 nL per well of compound diluted in DMSO. Cells were lysed in 60 µL of lysis buffer (20 mM Tris–HCl [pH 7.5], 0.5% Triton-X100, 150 mM NaCl, 1 mM EDTA, and 1 mM EGTA) per well for 1 h at room temperature. For the V5-xpress assay, anti-xpress (ThermoFisher) was used as capture at 2 µg/mL and SULFO-TAG labeled anti-V5 (Abcam #SV-Pk1) was used as detection at 0.25 µg/mL. For the full-length p53 MSD ELISA, anti-p53 (Abcam #ab59243) was used as capture at a 1:2000 dilution and SULFO-TAG labeled anti-p53 antibody from the Total p53 Whole Cell Lysate Kit (Meso Scale Discovery #K150DBD-1) was used as detection. MSD plates (96-well, MSD #L15XA-3/L11A-3) were coated with antibody overnight at 4°C, washed three times and blocked for 1 h at room temperature. A volume of 5 µL of detection antibody and 20 µL lysate per well were added and incubated 2 h at room temperature. Plates were washed three times, 150 µL of read buffer added, and plates were read on a SECTOR Imager 6000 (Meso Scale Discovery).