Nitrotyrosine

H9c2 cells and mice hearts were freshly harvested and homogenized in RIPA lysis buffer (Beyotime, Beijing, China), sonicated for 20s, and normalized with the BCA Protein Assay Kit (Beyotime). The protein was separated by 10% SDS/PAGE and then electro-transferred onto nitrocellulose membrane (Millipore, MA, USA). After blocked with 5% non-fat milk for 2hrs, the nitrocellulose membrane was washed in Tris-Buffered Saline Tween-20 (TBS-T) for three 10mins and incubated with gentle agitation at 4°C overnight with the primary antibodies to PARP-1 (1:2000, sigma, CA, USA), ICAM-1 (1:500; Santa Cruz Biotechnology, CA, USA), iNOS (1:1000, cell signaling technology, MA, USA), cleaved caspase-3 (1:1000, cell signaling technology), cleaved caspase-9 (1:1000, cell signaling technology), IGF-1R (1:1000, cell signaling technology), p-IGF-1R (1:1000, cell signaling technology), Akt (1:1000, cell signaling technology), p-Akt (1:1000, cell signaling technology), nitrotyrosine (1:2000, cayman, MI, USA), β-actin (1:1000, Cell Signaling Technology) followed by washes with TBS-T and subsequent incubation with appropriate horseradish peroxidase-conjugated secondary antibody for 2hrs at room temperature. Labeled protein was visualized through enhanced chemiluminescence (Millipore).

The primary antibodies used in this study included rabbit anti-4-HNE (HNE11-S, Alpha diagnostics, San Antonio, TX, USA), heme-oxygenase 1 (Enzo, Farmingdale, NY, USA), nitrotyrosine (10189540, Cayman Chemical, Ann Arbor, MI, USA), pGSK3β Ser9 (9322, Cell Signaling Technology, Danvers, MA, USA), GSK3β (32391, Abcam, Cambridge, MA, USA), pERK1/2 (9106, Cell Signaling Technology, Danvers, MA, USA), total ERK1/2 (9102, Cell Signaling Technology), pJNK (9251, Cell Signaling Technology), total JNK (9252, Cell Signaling Technology), p38 (Cell Signaling Technology), pp38 (Cell Signaling Technology), and actin (clone C4, EMD Millipore, Burlington, MA, USA). Antibodies specific for phosphorylated tau at sites Ser 262 (ab4856, Abcam), Ser202/Thr205 (AT8, Invitrogen, Waltham, MA, USA), and Ser396/Ser404 (PHF1, gift of Sharon Greenberg and Peter Davies), as well as the conformational tau-specific antibody Alz50 (gift of Peter Davies, Albert Einstein College of Medicine, Bronx, NY, USA) and total tau (tau 5, Invitrogen), were used. Secondary antibodies used in this study included an anti-mouse/rabbit HRP-linked secondary antibody (7076 and 7074, Cell Signaling Technology, Danvers, MA, USA) and a goat anti-mouse or goat anti-rabbit (AP124 or AP132, Millipore, Temecula, CA, USA) peroxidase-conjugated antibody.