Figure 5.

Celastrol binds adenylyl cyclase-associated protein 1 (CAP1) protein. (A) Schematic representation of the TRAP experiment. (B) Celastrol binding proteins using target-responsive accessibility profiling (TRAP) experiment. (C) and (D) Cells were incubated with celastrol or DMSO for 2 h, and cellular thermal shift assays (CETSA) analyzed the thermal stabilization of CAP1 protein at different temperatures and concentrations. (E) Chemical structure of the biotin labeled celastrol (Cel-biotin). (F) Cel-biotin was added to streptavidin-agarose beads and incubated. Biotin alone was used as a control. Lysates prepared from THP-1 cells were added to the streptavidin-agarose beads with Cel-biotin. Eluent was then loaded on a polyacrylamide gel for Western blot analysis. Total lysates were used as an input control. (G) Immunofluorescence staining of phorbol ester (PMA)-differentiated THP-1 cells were treated with celastrol for 2 h, and then stimulated with biotin or Cel-biotin for 4 h. (H) The interaction of celastrol with CAP1 or vector was measured by microscale thermophoresis (MST). The K_D value of celastrol and CAP1 interaction was determined with MO.Affinity Analysis Software. (I) Isothermal titration calorimetry (ITC) enthalpogram of the interaction between celastrol and CAP1 at 25 °C. The titration curve is depicted as a function of the molar ratio between CAP1 and the calculated concentration of celastrol in the assay. Data represent as mean ± SEM. P values are determined by two-tailed Student's t test (n = 3). ∗∗P < 0.01.