Antibodies against disulfidebond A oxidoreductase-like protein (DsbA-L) and CTRP9 were bought from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies against CHOP, GRP-78, TNF-[alpha], Caspase-3, Caspase-9, Caspase-12, and GAPDH were from Cell Signaling Technology (Beverly, MA, USA).
As depicted in Figure 2, western blot analysis of isolated perfused heart homogenates revealed that ERS was significantly increased in diabetic hearts after IR, as demonstrated by significantly increased ERS markers, glucose-regulated protein, 78 kD (GRP-78), and C/EBP-homologous protein (CHOP) expression compared with normal hearts (Figures 2(a), 2(c), and 2(d)).
TG treatment led to significantly increased ERS and inflammatory response, as demonstrated by enhanced CHOP, GRP-78, and TNF-[alpha] expression, together with greatly reduced cellular protein levels of DsbA-L (Figures 3(a), 3(b), 3(e), 3(f), and 3(g)).
DsbA-L knockdown induced ERS in H9c2 cells as shown by significantly increased CHOP and GRP-78 expression (Figures 4(a), 4(e), and 4(f)).
Representative images (a) and bar graphs of cardiac TNF-[alpha] (b), CHOP (c), GRP-78 (d), and DsbA-L (e) determined by Western blots.