Lc3b d11

Antibodies used for Western blot were purchased from Cell signaling technology, Danvers, MA, USA: AKT (pan) (C67E7), phospho-S473-AKT (D9E), phosphor-T308 AKT (244F9), AMPKα, phospho-T172-AMPKα (40H9), p70-S6K, phospho-T389-p70-S6K, S6P (5G10), phospho-S235/236-S6P, Raptor (24C12), Rictor (53A2), elF2α, phospho-S51-elF2α (119A11), ULK1 (D8H5), phospho-S757-ULK1, phospho-S555-ULK1 (D1H4), 4-EBP1 and LC3B (D11) XP. Antibodies used for immunocytochemistry: elF3η (C-5) was purchased from Santa Cruz Biotechnology, Dallas, TX, USA, and G3BP1 was purchased from Thermo Fischer Scientific, Waltham, MA, USA.

Dengue virus infection and inhibitor treatment experiments were performed with Huh-7 and A549 cells, and viral titers in the culture supernatants were determined by plaque assays on BHK-21 cells. IC₅₀ (50% inhibitory concentration) experiments were performed by using 2-fold dilutions of the inhibitor starting from an 8 μM concentration. A total of 30,000 cells were plated in a 48-well plate, infected with DENV-2 at a multiplicity of infection (MOI) of 3, and incubated with 2% Dulbecco's modified Eagle's medium (DMEM) containing inhibitors. Supernatants were collected at 24 h postinfection (p.i.), and viral titers were estimated by plaque assays on BHK-21 cells. For Western blotting, cell lysates were prepared as described previously (10 (link)), under the inhibitor treatment conditions described below, and the membranes were probed with antibodies recognizing DENV nonstructural protein 3 (NS3) (kind gift from Raj Bhatnagar), tubulin monoclonal (Developmental Studies Hybridoma Bank), calnexin (Thermo Fisher), and 78-kDa glucose-regulated protein (GRP78) (BD Biosciences) antibodies. For LC3 detection, an antibody that has a high specificity for the LC3-II form was used (LC3b [D11]; Cell Signaling Technology). Signals were detected by chemiluminescence. (See the supplemental material for a description of the flow cytometry experiment.)

Cells were lysed on ice in lysis buffer [phosphate-buffered saline (PBS), (pH 7.4), containing 1% Triton X-100] (Nacalai Tesque) and a protease inhibitor cocktail (Roche, Mannheim, Germany). Equal amounts of protein from each sample were heated to 95 °C for 5 min, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred to a polyvinylidene difluoride membrane (Millipore, Berlin, Germany). The membranes were blocked with 5% bovine albumin (Nacalai Tesque) and 1% or 5% non-fat dry milk (Cell Signaling Technology, Beverly, MA, USA) at 25 °C for 1 h. The membranes were then probed overnight with primary antibodies specific for H3K4me3 (Cell Signaling Technology), LC3B (D11) (Cell Signaling Technology), LSD1 (C69G12) (Cell Signaling Technology), phospho-PDH α1 (Ser293) (Cell Signaling Technology), PDH (Cell Signaling Technology), p62 (MBL Life Science, Nagoya, Japan), or β-actin (Sigma-Aldrich). Immunolabeled proteins were detected using horseradish peroxidase-conjugated secondary antibodies (GE Healthcare, Buckinghamshire, UK) and ECL Prime detection reagents (GE Healthcare). The signals were visualized using an ImageQuant LAS 4000 (version 1.3) system (GE Healthcare).