Anti rage

Adenovirus expressing β-galactosidase, APE1/Ref-1, or PPTLS-APE1/Ref-1 was prepared as previously described [24 (link)]. The effect of each adenovirus-mediated protein expression on cell viability and DNA fragmentation of MDA-MB-231, MDA-MB-468, and BT-549 cells was determined using a RealTime-Glo MT luminescent kit (Promega, Madison, WI, USA) and Cell Death Detection ELISA kit (Roche Applied Science, Indianapolis, IN, USA), respectively.
APE1/Ref-1 derived from cells expressing an adenoviral-mediated protein in the culture supernatant or whole-cell lysates was immunoprecipitated using anti-RAGE (Cell Signaling Technology, Beverly, MA, USA), followed by immunoblotting using anti-APE1/Ref-1 or anti-RAGE antibodies as described previously [19 (link)].

Immunoblot analysis was performed as previously described [22 (link), 23 (link)]. The following antibodies were used: anti-MGMT (1:1,000; Cell Signaling Technology, Danvers, MA, USA), anti-RAGE (1:1,000; Cell Signaling Technology, Tokyo, Japan), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:20,000; Trevigen, Gaithersburg, MD, USA), horseradish peroxidase-linked anti-rabbit IgG (1:20,000; GE Healthcare UK Ltd., Amersham Place, Little Chalfont, UK), and horseradish peroxidase-linked whole antibody anti-mouse IgG (1:20,000; GE Healthcare).

Western blotting was performed as previously described [22 (link)]. Anti-RAGE (1:1000, #6996S; Cell Signaling Technology, Inc., MA, USA), anti-STAT3 (1:1000, #12640; Cell Signaling Technology, Inc., MA, USA), anti-phosphorylated STAT3 (1:2000, #9145; Cell Signaling Technology, Inc., MA, USA), anti-cleaved caspase 3 (1:1000, #9664; Cell Signaling Technology, Inc., MA, USA), anti-LC3II/I (1:1000, #4108; Cell Signaling Technology, Inc., MA, USA), anti-Beclin1 (1:1000, #3495; Cell Signaling Technology, Inc., MA, USA) and anti-GADPH (1:1000, #2118; Cell Signaling Technology, Inc., MA, USA) were used as primary antibodies. The membranes were incubated with primary antibodies at 4℃ overnight, followed by incubation with HRP‐conjugated secondary antirabbit antibody (1:50,000; cat. no. BM2006; BOSTER Biological Technology Co., Ltd., Wuhan, Hubei, China) at 37 °C for 1 h after washing. anti-GADPH was used as an endogenous control for other proteins. Images were obtained using a chemiluminescent western blot scanner.