Enhanced chemiluminescence detection

Each sample was lysed in buffer containing 0.1% sodium dodecyl sulfate, 1.0% Nonidet-P40, 0.5% sodium deoxycholate, 50 mM Tris, pH 8.0, 150 mM NaCl, and a protease inhibitor (Roche Applied Science, Vienna, Austria, pH 7.4). Tissue samples were minced with scissors, and then the total protein was extracted. Electrophoresis was performed as described previously [45 (link)]. The primary antibodies involved included anti-SHMT2, anti-OCT4, anti-SOX2, anti-NANOG, anti-non-phospho-β-catenin, anti-β-catenin, anti-CYCLIN D1, anti-Slug, anti-Snail, anti-β-actin (1:1000; Cell Signaling Technology Inc., Danvers, MA, USA), anti-GAPDH, anti-E-cadherin, anti-N-cadherin, anti-NICD1, and anti-HES1 (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA). After incubation with the corresponding horseradish peroxidase-conjugated secondary antibodies (1:5000; Santa Cruz Biotechnology), immune reactive bands were visualized by enhanced chemiluminescence detection (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Supernatants harvested from infected Rhileki cells were centrifuged at 3,500 rpm and precipitated with 8% polyethylene glycol (PEG) 8000 (Merck, Germany) overnight in 4°C. The solution was centrifuged at 12,000 rpm for 1 h at 4°C, and pellets were resuspended in NTE buffer (1 M NaCl, 1 M Tris-HCl [pH 8.0], and 0.5 M EDTA [pH 8.0]). The suspension was subjected to a 20% sucrose cushion via ultracentrifugation using a SW41 rotor at 32,000 rpm for 2 h at 4°C. The pellet was resuspended in NTE buffer and reduced with 5× lane marker sample buffer (Thermo Fisher, USA) at 95°C for 10 min. Purified virions were separated on a 4 to 20% Mini-PROTEAN TGX stain-free protein gel (Bio-Rad, USA). The gel was transferred to a polyvinylidene fluoride membrane and blocked with 5% bovine serum albumin at room temperature for 1 h. This was followed by overnight probing with primary antibody at 4°C and incubation with anti-mouse or anti-rabbit horseradish peroxidase-linked IgG secondary antibodies for 1 h. Blots were visualized using a charge-coupled-device imager detector (Bio-Rad, USA) after enhanced chemiluminescence detection (Amersham, UK). The primary antibodies used include anti-229E nucleocapsid mouse monoclonal (Eurofins Ingenasa, Spain) and anti-229E spike rabbit polyclonal antibodies (Genscript, USA).

Cells were lysed in a buffer containing 150 mM NaCl, 1.0% nonidet-P40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris (pH 8.0) and a protease inhibitor cocktail (Roche Applied Science, Vienna, Austria, pH 7.4). Frozen tissue samples stored in liquid nitrogen were minced with scissors. Each sample was homogenized in a lysis buffer at a ratio of 1:20 w/v. After centrifugation at 13,000 rpm for 20 min, the supernatant was used to measure the total protein. Electrophoresis was performed as described previously [48 (link)]. The following primary antibodies were used for Western blot analysis: anti-GDF15 (1:1000; Abcam, Cambridge, UK), anti-phospho-AKT (Ser 473), anti-total AKT, anti-phospho-ERK, anti-total ERK, anti-phospho-SMAD2/3, anti-total SMAD2/3, anti-EGR1, anti-vimentin, anti-Snail, anti-β-actin (1:1000; Cell Signaling Technology Inc., Danvers, MA, USA), anti-N-cadherin, anti-E-cadherin and anti-GAPDH (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA). Following incubation with the corresponding horseradish peroxidase-conjugated secondary antibodies (1:5000; Santa Cruz Biotechnology), immune reactive bands were visualized by enhanced chemiluminescence detection (Bio-Rad Laboratories, Inc., Hercules, CA, USA).