Hemagglutinin

All chemicals were of analytical reagent grade and used without further purification. Double deionized (DI) water (18.6 MΩ) was used throughout the research work. The thiol terminal aptamer was purchased from Nzytech (Lisboa, Portugal) with a sequence SH-(CH₂)₆−CAG CAC CGA CCT TGT GCT TTG GGA GTG CTG GTC CAA GGG CGT TAA TGG ACA-3′ [14 (link)]. Syndrome coronavirus-2 receptor-binding domain (SARS-CoV-2-RBD), human serum albumin (HSA), human immunoglobulin A (HIgA), human immunoglobulin M (HIgM), human immunoglobulin G (HIgG), hemagglutinin (HA), neuraminidase (N), sodium tetrachloroaurate (NaAuCl₄), sodium borohydride (NaBH₄)_, phosphoric acid (H₃PO₄), potassium hydroxide (KOH), potassium ferricyanide (K₃Fe(CN)₆), potassium ferrocyanide (K₄Fe(CN)₆), chitosan, and hexaammineruthenium (III) chloride ([Ru(NH₃)₆]Cl₃) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dual working screen-printed electrodes (Ref X1110) were obtained from Metrohm-Drop Sens (Llanera, Spain).

Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (Carlsbad, CA, USA). Oil Red O, palmitic acid, low fatty acid bovine serum albumin (BSA), cycloheximide (CHX), GW7647, and MG132, as well as the anti-FLAG M2 affinity gel and antibodies against FLAG, hemagglutinin (HA), and GAPDH were obtained from Sigma-Aldrich (St. Louis, MO, USA). The antibody against FTO was from Abcam (Cambridge, MA, USA) and the anti-PPARα antibody was from ProteinTech Group (Rosemont, IL, USA). The secondary antibodies were all from Jackson Laboratories (West Grove, PA, USA).

MDCK cells were grown into 6-well plates and infected with H1N1 A/PR/8/34 virus for 2 h. The cells were then washed with PBS and the media was replaced with new media with various concentrations of the test compounds. After 24 h of incubation, the cells were washed with cold PBS and then lysed with 100 µL of lysis buffer [50 mM Tris-HCl (pH 7.6), 120 mM NaCl, 1 mM EDTA, 0.5% NP-40, and 50 mM NaF]. The supernatants were collected, and the protein concentrations were determined using a protein assay kit (Bio Rad Laboratories Inc., USA). The aliquots of the lysates were boiled for 5 min and loaded onto 10% or 12% SDS-polyacrylamide gels. The gels were then electrophoresed and electrotransferred to polyvinylidene fluoride membranes (PVDF 0.45 µm, Immobilon-P, USA). After blocking with a 5% skim milk solution, the membranes were incubated overnight at 4 °C with primary antibodies, namely, neuraminidase (Gene Tex, San Antonio, TX, USA), hemagglutinin (Sigma, St Louis, MO, USA), or mouse monoclonal actin (Abcam, Cambridge, UK). After washing a few times with PBS, the membranes were incubated with secondary antibodies for 2 h and then detected by a chemiluminescence Western blotting detection kit (Thermo Fisher Scientific., Rockford, IL, USA) using an Image Quant^TM LAS4000 imaging system.