2.6. Cellular Thermal Shift Assay (CETSA)

CETSA was performed as previously described (Jafari et al., 2014), with the following modifications. Briefly, cells were plated in cell culture dish using appropriate growth media and allowed to incubate overnight. Next day, regular growth media was replaced with culture media containing variable concentrations of DBeQ and ML240, for 2 h. Cells were trypsinized, and cell pellets were collected following centrifugation. Cell pellets were dissolved in PBS pH 7.0 and subjected to heat treatment at 57 °C. Following heat treatment, protein was precipitated using freeze‐thaw in liquid nitrogen. Cells were then subjected to centrifugation at 20,000×g, and soluble fractions were analyzed using Western blot. Blots were incubated with antibodies against VCP and β‐actin.