Hmgb1

Frozen testicular tissue was prepared with lysis buffer (150 mM NaCl, 20 mM Tris HCl [pH 7.4], 0.1% sodium dodecyl sulfate [SDS], 1.0% Nonidet P-40, 0.5% sodium-deoxycholate, 0.2 mM phenylmethylsulfonyl fluoride, protease inhibitor cocktails, and phosphates inhibitors). Equal amounts of proteins were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE), and electrotransferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The proteins were then blocked with 5% BSA for 2 h at room temperature. The membranes were incubated with appropriately diluted rabbit anti-rat primary antibodies for the detection of HO-1 (Abcam, UK), MFN-2 (Boshide, Wuhan, China), HMGB-1 (Santa Cruz Biotechnology Co., USA), and mouse anti-rat β-actin (Santa Cruz Biotechnology Co., USA) overnight at 4°C. The membranes were then incubated with the relevant secondary antibodies (HO-1, MFN-2, HMGB-1: anti-rabbit IgG, β-actin: anti-mouse IgG; all purchased from Boshide, Wuhan, China) for 2 h at 20°C. The proteins were detected with an enhanced chemiluminescence (ECL) detection system. β-Actin served as the internal control. The analysis was repeated thrice.

pCAGGS-HMGB1 (pHMGB1) and pCAGGS (empty vector) were kindly provided by Professor Tadatsugu Taniguchi (University of Tokyo, Tokyo, Japan) [40 (link)]. HMGB1 blocker glycyrrhizin was purchased from Sigma. glycyrrhizin was dissolved with PBS. TLR2/4 inhibitor OxPAPC was purchased from invivogen. RAGE-Fc was purchased from R&D Systems. The RAGE, HMGB1, and control siRNA were purchased from Santa Cruz Biotechnology. Macrophages were transfected with 200 nM of indicated siRNAs by Mouse Macrophage Nucleofector Kit (Lonza) according to the manufacturer's instructions. HMGB1 and RAGE antibody were obtained from Cell Signaling Technology and GAPDH antibody from Santa Cruz Biotechnology.

Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK). The membranes were probed with primary antibodies against RAGE and HMGB-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or β-actin (Sigma, St Louis, MO, USA).