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. Author manuscript; available in PMC: 2016 Aug 29.
Published in final edited form as: J Am Chem Soc. 2015 Jun 15;137(24):7929–7934. doi: 10.1021/jacs.5b04284

Figure 3.

Figure 3

BRD0476 interacts with USP9X in both β cells and cancer cells. (a) Log2 ratios of changes of protein abundance for cells incubated with immobilized compound in the absence or presence of soluble competitor. Proteins in INS-1E cells were metabolically labeled with light and heavy amino acids lysine and arginine using SILAC methodology. Cell lysates were incubated either with BRD0476-loaded beads and 30× soluble BRD0476 or BRD0476-beads alone. Each dot represents a distinct protein. (b) Cellular ATP levels in cytokine-treated INS-1E cells following knock-down with three individual siRNA constructs for Usp9x. (c) Caspase-3 activity in cytokine-treated INS-1E cells following knock-down with three individual siRNA constructs for Usp9x. (d) Immunoblot for USP9X and actin for INS-1E wild-type and CRISPR/Cas9-knockout clone. (e) Caspase-3 activity in INS-1E wild type (black bars) and CRISPR/Cas9-knockout clone (white bars) treated for 1 day with cytokines and BRD0476. (f) Cellular ATP levels in DLD-1 colon carcinoma cells treated for 48 h with the indicated concentration of the BCL2 inhibitor ABT-737 and 10 μM BRD0476. * p < 0.0001 compared to DMSO treatment, Student’s t test.