CHST3 protein, human

EIA was done as described previously for other viral antibody determinations [18 (link)]. For coronavirus-specific antibody EIA, 96-well microtiter plates (Nunc Maxisorp, Thermo Fisher Scientific) were coated with 50 µL of GST-N (2.0 µg/mL), RBD-mFc-8×His (4.2 µg/mL), and S1-mFc-6×His (3.0 µg/mL) in PBS at 4°C overnight. GST (0.7 µg/mL) and proMstn-mFc-6×His (4.2 µg/mL) proteins were used as negative control antigens. After coating, the plates were washed once with washing buffer (0.05% Tween-20 in PBS). Serum samples were inactivated at 56°C for 30 minutes, and 100 µL of 1:300 diluted serum specimens in sample buffer (5% swine serum [Biological Industries], 0.1% Tween-20 in PBS), were incubated for 2 hours at 37°C. Horseradish peroxidase-labeled anti-human IgG (1:8000 dilution; Dako), anti-human IgA (1:8000 dilution; Invitrogen), or anti-human IgM (1:4000 dilution; Dako) antibodies in sample buffer was added and incubated for 1 hour at 37°C. TMB One (3, 3’, 5, 5’-tetramethylbenzidine, Kementec Solutions) was used as a substrate and after 20 minutes incubation at room temperature, the reaction was stopped with 0.1 M H₂SO₄. The absorbance was measured at 450 nm (Victor Nivo, PerkinElmer). The absorbance for negative control antigens was subtracted from the respective sample absorbance and the results were expressed as EIA units using a pool of negative serum samples (given a unit value of 0) and a pool of highly positive serum samples (unit value of 100) as standards. The unit values for SARS-CoV-2 GST-N–based EIA were adopted to other HCoV GST-N–based EIAs because no confirmed negative and highly positive control samples were available for SARS, MERS, and low-pathogenic coronavirus N antibody assays.

The in vitro kinase assay was performed as previously described (Wang et al., 2019 (link)). A reaction mixture (18 μL) containing 0.40 μg of GST-CDPK-6xHis kinase, 5–10 μg of substrates of GST-fused peptides GLR3.6 or GLR3.7, and 2 μL of 10X buffer (200 mM Tris pH 7.5, 100 mM MgCl2, 10 mM EGTA, and 11 mM CaCl₂) was used. Next, 2 μL of 50 μM ATP solution (spiked with 2.5 μCi [γ-³²P] ATP) was added to initiate the kinase reaction at room temperature (24–28°C) for 10 min. The reaction was stopped by adding 5 μL of 4X sodium dodecyl sulfate (SDS) sample buffer. Samples were analyzed using 10% SDS-PAGE. The γ-³²P-labeled signals were normalized to the amount of protein determined using Coomassie brilliant blue-stained gels. The γ-³²P-labeled signals were detected using an image analyzer (GE Healthcare, Typhoon 9400).

Protein sources for enzyme assays included tissue extracts (rabbit lung, rat brain, rat lung) and recombinant GST-INMTs (rat, rabbit and human). Enzyme assays were conducted in 1.5 mL microcentrifuge tubes and contained approximately 50 µg protein (stock protein concentrations ranged from 4.1 to 6.9 mg/mL for rat brain, 10.6–14.6 mg/mL for rat and rabbit lung, and 0.75–1.05 mg/mL for recombinant GST-INMTs). Radiometric enzyme assays utilized 4 mM tryptamine (Sigma Aldrich) or NMT (Cayman Chemical) along with 2.3 nmol C¹⁴-SAM (specific activity 52.6 mCi/mmol, PerkinElmer Inc.) in a final reaction volume of 150 µL with sodium phosphate buffer. Assay components were combined and incubated at 37 °C for 60 min, and reactions were stopped by adding 1 mL ice cold 0.5 M borate buffer (pH 9.5). To extract reaction products, the reaction mixtures were transferred to 15 mL Falcon tubes with 6 mL of a 97:3 toluene:isoamyl alcohol (T:IAA) solution and tubes were vortexed for three bursts of 5 s each and centrifuged at 1,650 g for 3 min. For quantification, 4 mL of the T:IAA organic layer was added to scintillation vials containing 5 mL scintillation cocktail (Ultima Gold; PerkinElmer Inc.), and radioactivity was counted for 1 min on a Beckman LS Scintillation Counter. Tubes with substrate (tryptamine) omitted served as background controls. Specific activity (SA) was calculated by subtracting the average CPM of 3 background controls for each condition and dividing the resulting CPM value by the amount of protein (µg) used. Results are presented as averages of triplicate experiments in both brain and lung tissues for n = 6 rats per genotype, and as 6 replicate experiments for each GST-INMT condition. Non-radioactive assays were carried out for analysis via uHPLC-MS/MS analysis. These assays utilized 1 mM tryptamine or NMT with 33 µM SAM in a final reaction volume of 150 µL with sodium phosphate buffer. Assay components were combined and incubated at 37 °C for 60 min. Tubes with substrate omitted served as background controls. Reaction products were immediately extracted by adding 1 mL T:IAA and vortexed. 950 µL of the organic phase was transferred to a new 1.5 mL tube, and the organic solvent was evaporated on a bench top VacuFuge™ concentrator (Eppendorf) at room temperature for 2 h. The resulting products were resuspended in 1 mL 30% methanol, vortexed for 3 min, and analyzed via uHPLC-MS/MS (see below for “Methods”). Samples that had NMT as a substrate were further diluted tenfold in 30% methanol. Assays with tissue extracts were run in duplicate for each rat (n = 6 per genotype), and assays with GST-INMT extracts were run in triplicate for each condition, with empty vector GST transformations serving as background controls. Sample identities were blinded by L.C. for uHPLC-MS/MS analysis conducted by N.G.G.

The coding sequences of MKK5^T215D/S221D (MKK5^DD) (48 (link)), MPK3, MPK6, GRF4, and GRF4^S248A were cloned into the pGEX-4T-1 vector to add a GST tag. The plasmids were transformed into E. coli BL21, and the production of recombinant GST-MKK5^DD, GST-MPK3, GST-MPK6, GST-GRF4, and GST-GRF4^S248A was induced with 0.5 mM isopropyl β-d-1-thiogalactopyranoside at 16°C for 12 hours and then purified with Glutathione Sepharose 4 Fast Flow (GE Healthcare, Chicago, IL, USA). The in vitro kinase assay was performed as described previously (49 (link)). In brief, 0.1 μg of recombinant GST-MKK5^DD, 0.2 μg of GST-MPK3 or GST-MPK6, and 2 μg of GST-GRF4 or GST-GRF4^S248A were incubated in a 30-μl kinase reaction [10 mM MgCl₂, 1 mM adenosine 5′-O-(3-thiotriphosphate), 50 mM tris-HCl (pH 7.5), and 1 mM DTT] at 23°C for 30 min. Then, 20 mM EDTA and 1.5 mM p-nitrobenzyl mesylate (ABclonal, Wuhan, China) were added and incubated at 23°C for another 30 min, and 5× loading buffer was used to terminate the reaction. Anti-TPE (ABclonal, Wuhan, China), an antibody that specifically recognizes phosphorylated proteins (53 (link), 54 (link)), was used to determine whether MPK3 and MPK6 can phosphorylate GRF4 and GRF4^S248A, and the anti-GST antibody (TransGen, Beijing, China) was used to determine whether the loading quantity of each protein was consistent.