Horseradish peroxidase conjugated antibody

The S1, S2, M, N and E proteins (same with the protein in Section 2.7) of the SARS-CoV-2 Wuhan-Hu-1 strain were utilized to coat 96-well ELISA plates (Corning, NY, USA) at a concentration of 0.1 μg per well and incubated overnight at 4 °C. Diluted serum and standard samples were added to 96-well plates precoated with specific antibody. After washing, horseradish peroxidase-conjugated antibody (Thermo Fisher, Waltham, MA, USA) and 3,3′,5,5′-tetramethylbenzidine reagent (Solarbio, Beijing, China) were added successively for signal development. The stop solution was then added (Solarbio, Beijing, China), and the absorbance at 450 nm was read with a microplate reader. The specific IgG titers were determined by end titration utilizing the reciprocal of the lowest serum dilution that produced an OD value 2.1-fold greater than that in the prebleed. In addition, commercialized anti-SARS-CoV-2 (Omicron B.1.1.529) antibody IgG titer serologic assay kit was used to test the S1-specific IgG titers (Acrobiosystems, Newark, DE, USA) according to the manufacturer’s protocol.

To demonstrate production of ANME-1 Mcr, a FLAG epitope tag was introduced into the carboxy terminus of ANME-1 McrA encoded by mcrA in pES1-MATmcr1, pES1-MATmcr2, and pES1-MATmcr3. The DNA encoding the FLAG tag was incorporated into primer B6-r-flag, which was used along with the respective forward primers to create ANME-1 mcrA-flag. After transformation, M. acetivorans harboring pES1-MATmcr1-flag, pES1-MATmcr2-flag, and pES1-MATmcr3-flag were grown on 200 mL HS-methanol for 5 days, and used for short-duration growth experiments. Methane consumption was measured after 5 days, and cells were harvested by centrifugation. Each cell pellet was resuspended in 2 mL Lysis Buffer [20 mM Tris–HCl, 0.1 mM EDTA, 500 mM ε-aminocaproic acid, 10 % glycerol, 1 µL protease inhibitor cocktail (Sigma)]. Cells were sonicated on ice at a power level of 10 for 150 s (30 cycles of 5 s each, 60 Sonic Dismembrator, Fisher Scientific). Total proteins were resolved via 12 % Tris–glycine-SDS gels. Western blots were performed with monoclonal horseradish peroxidase-conjugated antibodies raised against a FLAG epitope tag (Thermo Scientific, Waltham, MA, USA). Blotted proteins were detected using the chemiluminescence reagents from the SuperSignal West-Pico Chemiluminescence kit (Thermo Scientific).

PIV5 that was grown in CHO-CD59 cells (PIV5-CD59) was pelleted by ultracentrifugation (25,000 RPM, 4 h, SW28 rotor) and then further purified on 20% to 60% sucrose gradients, which was detailed previously [32 (link)]. Fractions collected from the bottom of the tube were analyzed by Western blotting with rabbit serum specific for the PIV5 P protein or with rabbit anti-human CD59 antibody (Cell Signaling Technology, Danvers, MA, USA). In the case of CHO-CD59 cells, lysates were also probed with mouse anti-human actin to normalize loading amounts. To quantitate CD59 levels, dilutions of purified virus were analyzed by Western blotting along with known amounts of recombinant human CD59 protein (EMC Biochemical, Hopkinton, MA, USA). Blots were visualized by horseradish peroxidase-conjugated antibodies and chemiluminescent substrate (Thermo Scientific, Waltham, MA, USA).

Cells lysates (20–40 μg protein per sample) were separated under reducing conditions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad Laboratories, Hercules, CA) and transferred onto a polyvinylidene fluoride membrane (PerkinElmer, Waltham, MA) by semidry electroblotting (36 (link)). Membranes were probed with antibodies against human eNOS (BD Bioscience, San Jose, CA; Abcam, Cambridge, MA; Cell Signaling Technology, Danvers, MA) and phospho-eNOS (P-eNOS Ser-1177; BD Bioscience), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), SP1 and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA), A20 (Abcam), and β-galactosidase (Novus Biological, Centennial, CO), followed by the appropriate secondary horseradish peroxidase-conjugated antibodies (Thermo Scientific, Rockford, IL). Protein bands were detected with enhanced chemiluminescence kit (PerkinElmer, Waltham, MA) after exposure to an autoradiography film. The intensity of the scanned bands was quantified by densitometry using the ImageJ 1.41 software (NIH, Bethesda, MD). Alternatively, IRDye® infrared secondary antibodies were used and WB imaging was digitally acquired using the Odyssey® CLx imaging System. The intensity of the bands was quantified using the Image Studio^TM Software (Li-COR Inc, Lincoln, NE).