PA4 is a potent CAMKIIγ inhibitor. (A) Scheme of CETSA assay. (B) H9 cells were treated with DMSO or 5 μM PA4 for 1 hour. Next, the cells were collected in PBS and heated from 45°C to 52.5°C. Then, the proteins were extracted and subjected to western blot analysis of indicated proteins expression level. (C) The quantitative analysis of CAMKIIγ shown in panel B. (D) H9 cells were pretreated with 0, 5, 10, or 15 μM PA4 for 1 hour and then heated at 55°C. The western blot analysis of CAMKIIγ expression level. (E) The quantitative analysis of CAMKIIγ expression level from panel D. Data were expressed as mean ± standard deviation (SD), n = 3; ∗P < .05 and ∗∗P < .01 vs DMSO treatment. (F) The ratio of fluorescence 350:330 nm and the first derivatives of the transition curves from nano-DSF assay were plotted against the temperature. The melting temperature (Tm) values are derived from the maxima of the obtained first derivative curves. dTM, deviation of Tm.