Figure 5.

Dapagliflozin increases discoidin domain receptor 1 proteolytic cleavage. (a) Effect of dapagliflozin on DDR1 (N-terminus antibody). HCT116 cells were treated with either vehicle (−; lane 2) or 0.5 mM dapagliflozin (+; lane 1) for 20 min. Cell extracts were prepared and immunoblotted with an amino-terminal anti-DDR1 antibody. Only full length-DDR1 was detected. The quantification of the DRR1 protein is presented as a bar graph (n = 3, * p < 0.001). The closed column represents cells treated with 0.5 mM dapagliflozin and the open column represents DMSO-treated cells. The experiments were conducted independently in triplicate. (b) Effect of dapagliflozin on DDR1 (589G antibody). The immunoblotted membrane used in 5a was stripped and subsequently immunoblotted with 598 G antibody, which recognizes a proximal transmembrane domain epitope. Full length-DDR1 and cleaved-DDR1 showed up. (c) Effect of dapagliflozin on DDR1 (C-terminus antibody). The immunoblotted membrane used in Figure 5a,b was stripped and immunoblotted with a carboxy-terminal anti-DDR1 antibody. Full length-DDR1 and cleaved-DDR1 showed up as Figure 5b. (d) Estimation of loading amount. The immunoblotted membrane used in 5a–c was stripped and immunoblotted with anti-alpha-tubulin antibody. (e) Estimation of sheddase activity with or without dapagliflozin. Proteolytic activity was determined as described in the Experimental procedures section (n = 3, * p < 0.05). In brief, specific substrate, and dapagliflozin were mixed with the buffer and incubated for 60 min at room temperature. The resulting fluorescence was measured at 520 nM. Open column represents that the proteolytic activity was measured with 0.5 mM dapagliflozin and closed column represents that the proteolytic activity was measured with DMCO.