Pierce coomassie plus assay kit

Protein samples from the transfected cells were prepared in 1× SDS gel-loading buffer (Laemmli buffer) without adding β-mercaptoethanol and quantified using the Pierce™ Coomassie Plus Assay kit (Thermo Scientific, Rockford, IL, USA). Without heat denaturation, equal amount of protein samples (50 μg) were loaded on a 10% SDS-polyacrylamide gel. After electrophoresis, the gel was incubated in NBT/BCIP (Roche, Mannheim, Germany) staining solution until the bands corresponding to ALPL were clearly visible. Same protein extracts upon heat denaturation in the presence of β-mercaptoethanol were used for Western blotting. The primary antibody used for the analysis was anti-actin (Sigma-Aldrich, Saint Louis, MO, USA). An anti-actin antibody raised in rabbit anti-actin (Sigma-Aldrich, Saint Louis, MO, USA). The secondary antibody was anti-rabbit HRP-IgG (Cell Signaling Technology, Beverly, MA, USA).

Pig hearts were obtained after euthanasia and placed in a cardioplegic solution (280 mM glucose, 13.44 mM KCl, 12.6 mM NaHCO₃, and 34 mM mannitol). Left ventricles free walls were obtained, minced, and homogenized with a cold buffer that contained 9.1 mM NaHCO₃, 0.9 mM Na₂CO_3, and a cocktail of proteases inhibitors (Sigma); the mixture was centrifuged at 6,500 × g for 30 min at 4 °C to remove debris. The supernatant was filtered, collected, and centrifuged at 14,000 × g for 30 min at 4 °C. The collected filtrate was centrifuged at 47,000 × g for 60 min at 4 °C. The pellet was resuspended in a solution containing 0.6 M KCl and 20 mM Tris (pH = 6.8). The suspension was centrifuged at 120,000 × g for 60 min at 4 °C, and the pellet was resuspended in a solution containing 0.3 M sucrose, 5 mM 3-(N-Morpholino)propanesulfonic acid (MOPS), and protease inhibitors (pH = 7.4). The protein concentration of the SR microsomal fraction was determined using the Pierce Coomassie plus assay kit (Thermo-Fisher Scientific, Waltham, MA, USA). The microsomal membranes were aliquoted, frozen in liquid nitrogen, and stored at −80 °C. SERCA2a purification was performed as described by Sitsel et al. (29 (link)).

Cells grown on a 6-well plate were infected with various rMeVs at an MOI of 0.03. At 36–48 h post-infection, the supernatant was collected and filtered through a 0.45-μm pore membrane. Additionally, the cells were lysed in mammalian protein extraction reagent (Cat# 78503, Thermo Fisher Scientific) supplemented with Halt protease inhibitor cocktail (Cat# 877886, Thermo Fisher Scientific). The protein concentration was determined using a Pierce Coomassie Plus Assay Kit (Cat# 23236, Thermo Fisher), and 3 µg of cell lysate or ~20 µL of supernatant was separated on a precast 12% or 4%–12% Bis-Tris polyacrylamide gel before being transferred to a polyvinylidene fluoride membrane using an iBlot2 dry blotting system (Thermo Fisher Scientific). The blot was then probed with anti-SARS-CoV-2 spike RBD (GTX135385, GeneTex, Irvine, CA, USA), anti-SARS-CoV-2 spike (Cat# GTX632604, GeneTex), anti-MeV nucleocapsid (Cat# LS-C144599, LsBio, Seattle, WA, USA), anti-MeV hemagglutinin (116 (link)), anti-canine distemper virus fusion (Genscript), and anti-high affinity (HA) peroxidase (Cat#12013819001, Millipore Sigma, St. Louis, MO, USA) and developed with a KwikQuant Western Blot Detection Kit using a KwikQuant Imager (Kindle Bioscience LLC, Greenwich CT, USA).

HEK293T cells were plated onto poly-l-lysine–coated 24-well plates (Sigma-Aldrich, P6282) and grown to 50%-60% confluence. Cells were transfected with Flag-GLYT1 WT, Flag-GLYT1 variants, and pCMV-3Tag-1A backbone. The detailed procedure of glycine uptake assay was described previously (84 (link)). Briefly, prior to uptake, the cells were washed 3 times with assay buffer containing 116 mM NaCl, 1 mM NaH₂PO₄, 26 mM NaHCO₃, 1.5 mM MgSO₄, 5 mM KCl, 1.3 mM CaCl₂, and 5 mM glucose, and then incubated for 10 minutes with 1 μCi/mL [³H] glycine (60 Ci/mmol, PerkinElmer, NET004001MC) at a final concentration of 200 μM at 37°C. Glycine uptake was terminated by quick washing with ice-cold assay buffer followed by aspiration twice. Cells were digested in 0.1M NaOH, and the supernatants were subjected to scintillation counting (LS6500, Beckman Coulter) and protein concentration measurement using Bradford reagent (Pierce Coomassie Plus Assay Kit, Thermo Fisher Scientific, 23236). [³H]-glycine uptake was calculated as nanomoles per minute per milligram of protein (nmol/min/mg protein) and normalized as a percentage of that in control cells transfected with WT plasmid.

NF-κB activity was evaluated by measuring p65 translocation into the nucleus using a TransAM NF-κB p65 Kit (Active Motif, Carlsbad, CA) according to the manufacturer’s instructions. Nuclear extracts were extracted from cultured peritoneal macrophages, and protein concentrations were determined with a Bradford-based assay (Pierce Coomassie Plus Assay Kit, Thermo Fisher Scientific Inc.). The nuclear extracts were added to a 96-well plate coated with an oligonucleotide containing the NF-κB consensus sequence (5’-GGGACTTTTCC-3’) and incubated for 1 h at room temperature. An anti-NF-κB antibody was added to each well, and the plate was incubated for 1 h at room temperature. After the plate was incubated for 1 h with a horseradish peroxidase-conjugated secondary antibody, specific binding was detected with a microplate spectrophotometer based on the absorption at 450 nm with a reference wavelength of 655 nm.

Escherichia coli MET1158 strain, E. coli LW7 pLW11 and E. coli pBAC-LacZ were donated by Prof Karina Xavier (Instituto Gulbenkian de Ciência, Portugal), Prof. William Bentley (University of Meriland, USA) and Keith Joung (Addgene plasmid # 13422), respectively.
All chemicals were purchased from Sigma (USA) if not otherwise stated. DPD was acquired from Carbosynth (Compton, Berkshire, UK). PD-10 desalting columns and Protino^® Ni-NTA columns (1 mL) for protein purification were purchased from GE Healthcare Lifescience (Chicago, IL, USA) and Macherey-Nagel (Düren, Germany), respectively. Pierce Coomassie Plus Assay Kit was obtained from Thermo Fisher Scientific (Waltham, MA, USA). ATP Bioluminescence CLS II kit was purchased from Roche Diagnostics GmbH (Basel, Switzerland), Kinase-Glo Max Luminescent kinase assay kit from Promega Corp. (Madison, WI, USA) and ADP-Quest kit from DiscoveRx Corp. (Fremont, CA, USA). PopCulture™reagent and rLysozyme™ were purchased from Millipore (Burlington, MA, USA). Plates were purchased from Greiner Bio One (KremsMünster, Austria), for assay development and screening campaign, and from Thermo Fisher Scientific (Waltham, MA, USA) for AI-2 QS interference assay.