Antibody against hmgb1

Hypericin was prepared/purified/stored as described in the past [64 (link)]. Tauroursodeoxycholic acid (TUDCA) was purchased from Merck-Millipore (Darmstadt, Germany) while Z-Val-Ala-Asp(OMe)-fmk (zVAD-fmk) was purchased from Bachem (Weil am Rhein, Germany). Mitoxantrone (MTX) and Anti-actin antibody was purchased from Sigma (St. Louis, MO, USA). Topotecan and paclitaxel were kind gifts from Prof. Johan Swinnen/Niamat Ali Khan (KULeuven, Belgium). Antibodies against CHOP/P-eIF2α/Total eIF2α/cleaved caspase 3/PARP/HSP90/Bip/GRP78 were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-calreticulin antibody (for immunoblotting) was purchased from Stressgen (Victoria, BC, Canada). Antibody against HMGB1 was purchased from Abcam (Cambridge, UK). Anti-HSP70 antibody was purchased from Santa Cruz Antibodies (San Francisco, USA). Secondary antibodies conjugated to horseradish peroxidase were purchased from Cell Signaling Technology (Danvers, MA, USA)/Abcam (Cambridge, UK). Also, the following secondary antibodies were used: goat anti-mouse-DyLight680/goat anti-rabbit-DyLight800 (Thermo Scientific, Belgium).

Total protein was extracted from homogenized LV tissues and cultured cardiac myocytes using RIPA lysis buffer (Beyotime, Nanjing, China), culture supernatants were concentrated using Amicon Ultra Centrifugal Filter Devices (Millipore, Billerica, MA, USA) according to the manufacturer's protocol. Proteins were electrophoresed in 12% polyacrylamide gel and transferred to a polyvinylidene fluoride membrane (Millipore). The protein expressions was detected by immuneblotting with antibody against HMGB1 (Abcam). After three washes, the blot was incubated with horse radish peroxidase‐conjugated rabbit secondary antibody immunoglobulin G (Kangchen Biotechnology, Shanghai, China). β‐actin (Kangchen Biotechnology) was used as the internal control. The antigen–antibody complexes were detected using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Rockford, IL, USA) and visualized densitometry was performed with LAS‐300 Image software (FUJIFILM, Kanagawa, Japan).