Anti optineurin

RGCs were lysed using RIPA buffer (Beyotime Biotechnology, Shanghai, China) to obtain protein samples. Each sample (20 μg) was separated by SDS-PAGE gel and transferred to PVDF membranes. The membranes were incubated with several primary antibodies such as anti-parkin (1:200; Abcam), anti-optineurin (1:200; Abcam), anti-OPA1 (1:1000; Abcam), anti-LC3 (1:2000; Abcam), anti-LAMP1 (1:1000; Abcam), anti-USP30 (1:1000; Affinity), anti-MFN2 (1:1000, Abcam), anti-Ubiquitin Antibody (1:1000, LifeSensors) and anti-GAPDH (1:2000; Abcam) at 4 °C overnight and finally with peroxidase-conjugated anti-rabbit IgG (1:5000; Jackson). Images were developed with a chemiluminescence detection kit (SuperSignal™, Thermo Fisher) and analyzed by a 4000 MM PRO Kodak Image Station (Carestream, Rochester, NY, USA).

The following primary antibodies were used in this study: anti-PIWIL1 antibody (ab12337; Abcam, Cambridge, UK), anti-p-mTOR antibody (ab109268; Abcam), anti-LC3 I/II (#2775S; Cell Signaling Technology, Danvers, MA, USA), anti-P62/SQSTM1 (#8025S; Cell Signaling Technology), anti-Parkin (#4211; Cell Signaling Technology), anti-phosphor-NAK/TBK (ab109272; Abcam), anti-NAK/TBK (A5497; Bimake), anti-optineurin (ab213556; Abcam), anti-β-actin (#4970; Cell Signaling Technology), anti-Akt (#4691; Cell Signaling Technology), anti-phospho-Akt (Ser473) (#9271; Cell Signaling Technology), anti-Ki67 (#4685; Cell Signaling Technology), anti-Bcl-2 (A5010; Bimake), anti-OCT4 (60242-1-Ig; Proteintech), and anti-Nanog (#4903T; Cell Signaling Technology). Goat anti-mouse and goat anti-rabbit antibodies (Biosciences) were used as the secondary antibodies for western blotting. Dexamethasone and doxorubicin were purchased from Sigma (St. Louis, MO, USA), and bortezomib was purchased from Selleck Chemicals. 3-Methyladenine (3-MA) and the mitophagy inhibitor cyclosporin A (CsA) were purchased from MedChemExpress (NJ, USA). All were dissolved in dimethyl sulfoxide (DMSO) and stored at −20°C for up to 6 months. For all cell-based experiments, drugs were diluted at least by 1:1000 to ensure that the final DMSO concentration was lower than 0.1%.