Fig. 6. Off-target validation by PRM assay. (A) Workflow for the validation of off-targets via PRM assay that performed in single temperature TPP and ITDR modes. (B) Ring chart displaying the percentages of off-target validation for vemurafenib, palbociclib, alisertib, dinaciclib, and ralimetinib. (C and D) PRM-MS quantification of the selected potential off-targets. (C) K562 cell lysates were treated with a 20 μM drug or vehicle, followed by thermal treatment at 52 °C. (D) ITDR with treatment of eight concentrations (100, 27, 7.3, 2.0, 0.53, 0.14, 0.039, and 0.010 μM) of drug and vehicle, followed by thermal treatment at 52 °C. (E) NanoBRET analysis of palbociclib-PIP4K2C interaction in HEK 293T cells. A known inhibitor (UNC3230) of PIP4K2C was used as the positive control, and alisertib was taken as the negative control. n = 4 biologically independent replicates. (F) Western blot based ITDR for GRK2 at 52 °C. The band intensities were related to the intensities of the DMSO vehicle control samples. GAPDH levels were used to normalize the intensities. Data are reported as mean ± SD of three independent experiments.