Anti g6pd

Upon 24 h of infection and treatment, A549 cells were collected and lysed in cold RIPA buffer [20 mM Tris–HCl pH 8, 150 mM NaCl, 1% Triton X-100, 0.5% SDS and 1% sodium deoxycholate] supplemented with phenylmethyl-sulphonyl fluoride, protease inhibitor mixture, and phosphatase inhibitor (Sigma Aldrich, St. Louis, MO, USA). After 30 min of incubation, the cell lysates were centrifuged (14,000× g, 30 min, 4 °C). The supernatants obtained from the centrifugation were collected, and the protein concentration was determined by a Bradford protein assay. For the SDS-PAGE electrophoresis procedure, cell lysates samples were prepared by adding sodium dodecyl sulfate (SDS) buffer and DL-dithiothreitol. After the protein transfer, the nitrocellulose membrane was blocked with 10% milk solution (Bio-Rad Laboratories, Berkeley, CA, USA) diluted in T-TBS (0.01% Tween 20 plus Tris-buffered saline) for 1 h at room temperature and then incubated with a polyclonal goat anti-influenza antibody (anti-Flu, AB1074 Merck Millipore, Darmstadt, Germany), anti-Nrf2 (#12721 Cell Signaling) or with anti G6PD (#12263 Cell Signaling) overnight at 4 °C. After three washes with T-TBS, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody and developed using Clarity Western ECL substrate (Bio-Rad, Hercules, CA, USA). Actin was used as a loading control.

Cell were lysed with RIPA buffer and subjected to SDS-Page and immunoblotting according to standard methods essentially as described [8 (link),19 (link)]. Primary antibodies were anti-MFN2 mAB (1:500; Abnova, Taipei City, Taiwan), anti-G6PD (1:1000; Cell Signaling, Danvers, MA, USA), anti-PKM1/2 (1:1000; Cell Signaling), anti-Hexokinase I (1:1000; Cell Signaling), anti-Hexokinase II (1:1000; Cell Signaling) and anti-Actin mAB (1:4000; Merck Millipore, Burlington, MA, USA). Protein bands were revealed and analyzed following incubation for 1 h at room temperature with a secondary goat anti-mouse IgG antibody conjugated to an infrared fluorescent dye (IRDye 800, Licor, Bad Homburg, Germany) using the Odyssey near infrared laser imaging system (Licor, Bad Homburg, Germany). For mitophagy induction, cells were treated with 10 µM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) for the indicated time under normal culturing conditions. The reaction was stopped by washing steps with PBS and cell lysis with RIPA buffer.