Rabbit anti lc3a b

Primary antibodies used in this study: rabbit anti-LC3A/B, rabbit anti-Parkin rabbit anti-HSP60 (1:1000, Cell Signaling, products #4108S, #4211S and #12165S , respectively); mouse anti-TIM23 (1:1000, BD Biosciences, product #611222); mouse anti-VDAC1, mouse anti-TOM20, and mouse anti-p62 (Santa Cruz, 1: 500, sc-58649, sc-17764, and sc-28359, respectively); rabbit anti-ATP synthase gamma (1:1000, GeneTex, product #GTX-114275), mouse anti-Actin (1:3000, Genescript, product #A00702) and rabbit anti-GFP (Genescript, product #A01388-40). Secondary antibodies were: HPR conjugated goat anti-rabbit and goat anti-mouse (1:2500, products #170-6515 and #172-1011, respectively). CCCP and DMSO were obtained from Sigma. Bafilomycin A1 was obtained from LC Laboratories. Mitotracker Deep Red FM, Lysotracker Red DND 99, and TMRE, and Hoechst 33342 were obtained from Life Technologies. 3-Hydroxy-1,2-dimethyl-4(1H)-pyridone was from Sigma.

Cells grown on coverslips were fixed with 3.7% formaldehyde in PBS, 10 min at room temperature, followed by permeabilisation in PBS containing 0.1% TritonX-100, 5 min at room temperature. Blocking and incubations with secondary antibodies were performed at room temperature in PBS containing 0.05% Tween and 3% BSA. Primary antibodies used were: mouse anti-TOMM20 (Santa Cruz Biotechnology, sc-17764), rabbit anti-LC3A/B (Cell Signalling Technologies, 12741S). Cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI, 0.1 μg/ml; Sigma-Aldrich) and mounted using Flouromount Mounting Media (Sigma-Aldrich, F4680). Samples were analysed using a Zeiss LSM 800 microscope equipped with 63X or 100X (oil immersion) objectives. Images were acquired using ZEN system (ZEISS, Germany). ImageJ software was used for image analysis. Calculation of the mitochondrial content as percentage of cell area occupied by mitochondria, was performed using the “Mitophagy” macro [40 (link)].

All SiNPs treated HCECs were lysed in ice-cold RIPA buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, and 0.1% SDS) for 30 min. The debris was removed by centrifugation at 16,000 g for 1 min. Equal amounts (20 μg) of total cell protein were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a PVDF membrane. After blocking with 5% BSA in TTBS buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 0.1% Tween20) for 1 h at room temperature, membranes were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-LC3A/B (1:1,000; catalog number: 12741; Cell Signaling, Beverly, MA, USA), rabbit anti-phospho-mTOR (1:1,000; catalog number: 5536; Cell Signaling), rabbit anti-mTOR (1:1,000; catalog number: 2983; Cell Signaling) and mouse anti-β-actin (1:10,000; catalog number: sc-47778; Santa Cruz, Biotechnology, Dallas, Texas, USA). The membranes were incubated with peroxidase-conjugated secondary antibody for 1 h at room temperature. Blots were developed using an enhanced chemiluminescence (ECL) kit (catalog number: RPN2232; GE healthcare, Buckinghamshire, UK) and visualized using Fujifilm Image Reader LAS-3000 (Fujifilm, Tokyo, Japan). Each experiment was repeated at least 3 times, and densitometric analysis was performed using the Multi Gauge V3.0 (Fujifilm Life Science, Tokyo, Japan).

Human NSCs and differentiated neuronal cells were seeded on chamber slides. The cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.5% Triton X-100 in TBS for 5 min at room temperature. TBS with 2% FBS was used for blocking. The cells were incubated with the following primary antibodies at 4 °C overnight: rabbit anti-SOX2 (1:200, Cell Signaling, CA, USA), mouse anti-nestin (1:200, Millipore, MA, USA), rabbit anti-MAP2 (1:200, Millipore, MA, USA), mouse anti-Tuj1 (anti-neuron-specific class III β-tubulin, 1:200, Millipore, MA, USA), mouse anti-GAD67 (1:200, Millipore, MA, USA), rabbit anti-active caspase 3 (1:200, Millipore, MA, USA), rabbit anti-LC3A/B (1:200, Cell Signaling, CA, USA), mouse anti-EV-A71 3D (1:500, Genetex, CA, USA) or rabbit anti-EV-A71 3A (1:500). The cells were then washed with TBS and incubated with the following secondary antibodies for 1 h at room temperature: DyLight 488-conjugated goat anti-rabbit secondary antibody or DyLight 594-conjugated donkey anti-mouse secondary antibody (1:1,000, Jackson ImmunoResearch Laboratories, Pennsylvania, USA). The cells were then washed with TBS, and cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, MO, USA). Images were collected with an Olympus BX51 fluorescence microscope (Olympus, Tokyo, Japan) and an LSM 510 microscope (Zeiss, Jena, Germany).

Immunofluorescence was performed as described [65 (link)], using the following antibodies: goat anti-GFP FITC conjugated (1:400, Abcam), rabbit anti-LC3A/B (1:1000, Cell Signaling), mouse anti- SQSTM1/p62 (1:100, Novus Biological), rabbit anti-TFEB (1:100, Cell Signaling), rabbit anti-CathD (1:100, Calbiochem) and rabbit anti-Atg8a (1:200, Abcam). Anti-rabbit and anti-mouse Alexa Fluor secondary antibodies (Invitrogen) were used at 1:1000. Nuclei were visualized by staining the DNA with DAPI or Hoechst 33324 (Invitrogen). Images were acquired using a Leica TCS SP5 confocal microscope. Live cell images were obtained as previously described [18 ].