FIGURE 1.

Identification of FL118 biochemical targets. Detailed methods are described in the Materials and methods section. (A) FL118 affinity column purification identified a ∼70 kD protein. FL118 was directly coupled to a beaded resin. Then, SW620 protein lysates were purified with the FL118 affinity column through steps of stringent washing, elution with 8 M urea, de‐urea, sample concentration to 20–30 μl, which was displayed on 5%–20% SDS polyacrylamide gel electrophoresis gel. (B) The ∼70 kD protein in (A) was analysed via mass spectrometry. The protein band was digested in gel, and 10 peptides were isolated and used for searching the protein database. The 10 peptides fully matched the DDX5 (also called p68) protein sequence. (C), (D), (E), (F) Representative isothermal titration calorimetry (ITC) analysis results are shown. The K_D values presented within the figure are the mean ± standard deviation (SD) from two ITC analyses. The y‐axis, with different labelling scales, is for result pattern visibility and dot inclusion. Purified Flag‐tagged DDX5 (Flag‐DDX5) (C, F), Flag‐Top1 (D) and bovine serum albumin (E) were loaded into a 96 DeepWell PP plate, and then FL118 (C, D, E) or topotecan (F) was automatically titrated stepwise into the protein cell by 20 injections in 60 min (one injection per 3 min) on a MicroCal‐Malvern Auto‐ITC200