Anti flag tag m2 antibody

DF-1 monolayers in 6-well dishes at 90% confluence were inoculated with H7N7-LP_FLAGtag or H7N7-HP_HAtag at an MOI of 0.1 (TCID₅₀). After 1 h of incubation, cells were washed thrice with phosphate buffered saline (PBS) and overlaid with DMEM containing 0.08 μg/mL TPCK-treated trypsin without FCS. At 24 hpi, cells were trypsinized, and fixed and permeabilized with BD Cytofix/Cytoperm (BD Biosciences) according to manufacturer’s instructions. Cells were subsequently stained for 1 h on ice with the following antibodies diluted in BD Perm/Wash buffer: 20 μg/mL anti-influenza NP antibody (H16-L10-4R5 (ATCC HB-65)), followed by 10 μg/mL Alexa Fluor488-conjugated goat anti-mouse IgG2α antibody (A-21131; Thermo Fisher Scientific); 20 μg/mL anti-FLAGtag antibody (M2; Sigma Aldrich), followed by 10 μg/mL Alexa Fluor488-conjugated goat anti-mouse IgG antibody (A11029; Thermo Fisher Scientific); 7 μg/mL FITC-conjugated anti-HAtag antibody (H7411; Sigma Aldrich). After each staining, cells were washed twice with BD Perm/Wash buffer. Cells were subjected to flow cytometry on the FACSLyric (BD Biosciences) and the data were analyzed using FlowJo v10.7.2 software (BD Biosciences).

Protease and phosphatase inhibitors were purchased from Roche Diagnostics (Basel, Switzerland). Resveratrol and N-acetylcysteine (NAC) were procured from Wako Pure Chemical Industries, Ltd (Osaka, Japan). Acetoxy-forms of HCAs (PhIP: molecular weight 286.3; MeIQx: molecular weight 275.2) were synthesized (NARD Institute, Amagasaki, Hyogo, Japan). FICZ is a tryptophan photoproduct (Enzo Life Sciences, NY, USA). GW6471 PPARα inhibitor was purchased from Wako (Osaka, Japan). Neomycin (Wako) and hygromycin (Hygro, Sigma, MO, USA) were used after checking the cytotoxic effects in each cell line. Antibodies against PPARα (Abcam, Tokyo, Japan), sirtuin 1 (SIRT1), sirtuin 3 (SIRT3), sirtuin 6 (SIRT6), sirtuin 7 (SIRT7), p38, phosphorylated p38, CREB, phosphorylated CREB, and SNF2H (Cell Signal Technology, Tokyo, Japan), H2AX (Millipore), enhanced green fluorescent protein (MBL, Nagoya, Japan), Anti-FLAG Tag M2 antibody (Sigma MO, USA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Trevigen, Gaithersburg, MD, USA) were used as primary antibodies. A rabbit polyclonal antibody against human ORF1 was generated using the peptide MGKKQNRKTGNSKTQSAC as an immunogen (Medical and Biological Laboratories)^{14 (link)}. Anti-mouse and anti-rabbit secondary IgG conjugated to horseradish peroxidase were obtained from DAKO Japan (Tokyo, Japan).