Cellular Thermal Shift Assay (CETSA)

Thirty million K562 cells transduced with HMD control, BCL11A WT, and Mutant 1–4 lentiviral constructs were harvested by centrifugation at 300 r.c.f. for 10 minutes and 4°C, and washed twice with PBS, pH 7.4. The cells were resuspended at 30 x 10⁶ cells/mL in PBS containing PMSF. Each cell line was aliquoted into PCR tubes at 100 μL. Individual aliquots of cells were heated to 27°C, 37°C, 47°C, 57°C, and 67°C for 3 minutes. Cells were then cooled at room temperature for 3 minutes, and frozen at -80°C. Lysates were prepared by 3 freeze-thaw cycles, followed by vortexing. Cellular debris and protein aggregates were pelleted, and 90 μL supernatant was collected in fresh tubes for immunoblot analysis.