Cardiolipin

ELISA 96-well plates (Costar, Corning, NY, USA) were coated with 50 μL per well of cardiolipin (Sigma, St. Louis, MO, USA) prepared at 200 μg/mL in ethanol and blocked for 2 h with 4% BSA in a 20 mM Tris, 100 mM NaCl buffer at pH 7.4. To generate a binding curve, increasing concentrations of HA-DV were applied to wells. The binding data were fit to a one-site model using the Gnuplot 5.0 program (http://www.gnuplot.info/). cardiolipin binding by HA-DV variants was compared to cardiolipin binding by HA-DV at protein concentrations of 500 nM and 1000 nM. Bound HA-tagged proteins were detected with HRP-conjugated anti-HA-tag antibody (ab1265, Abcam, Cambridge, MA, USA) using a TMB (3,3′,5,5′-tetramethylbenzidine) substrate. Absorbances at 450 nm were measured on a Spectramax 340PC Microplate Reader (Molecular Devices Inc., Sunnyvale, CA, USA).

L-[methyl-³H]carnitine and L-[2,3-³H]ornithine from Scopus Research BV Costerweg, Sephadex G-75, egg-yolk phospholipids (l-α phosphatidylcholine from fresh turkey egg yolk), PIPES, HEPES, Triton X-100, cardiolipin, L-carnitine, L-ornithine, sodium itaconate, dimethyl itaconate (DMI), N-ethylmaleimide (NEM) were purchased from Sigma-Aldrich, Milan, Italy. All other reagents were of analytical grade.

The SP-B^N peptide was provided by Professor Timothy Weaver (Cincinnati Children′s Hospital Medical Centre, Cincinnati, OH, USA). The fluorescent dyes DPH, Sytox Green, DiSC₃-(5), ANTS, and DPX were from Molecular Probes (Eugene, OR USA). Rough lipopolysaccharide (Re 595, Re-LPS) and smooth lipopolysaccharide (S-LPS) from Salmonella enterica serotype Minnesota, phosphatidylethanolamine, and cardiolipin were from Sigma-Aldrich. Palmitoyloleoylphosphatidylglycerol and POPE were from Avanti Polar Lipids (Birmingham, AL, USA). The organic solvents used to dissolve the lipids were of HPLC grade.

For the polyreactivity ELISA, microplates (Immunoplates, Corning, USA) were coated with 10 μg/mL of double-stranded DNA (dsDNA) (Sigma-Aldrich), Sm (Meridian), nucleosome (Meridian), or cardiolipin (Sigma-Aldrich) in coating buffer (pH = 7.6) at 4°C overnight. Coated plates were blocked with 100 μL PBST/BSA buffer (2.0% BSA, 0.05% Tween-20) for 2 h at RT, followed by incubation with 100 μL of purified antibodies (1:4 serially diluted in PBS with 2% BSA) at RT for 1 h. After washing four times with PBST, horseradish peroxidase (HRP)-labeled goat anti-human IgG (H + L) antibodies (Proteintech, USA) were added 100 μL (1:3,000 dilution) to each well and incubated at RT for 1 h. For color development, 3,3′,5,5′-tetramethylbenzidine (TMB, Sigma-Aldrich) was added and incubated for 10 min. The reaction was stopped with 2 M H₂SO₄, and the absorbance at 450 nm (OD₄₅₀) was measured on a Biotek plate reader (Biotek, USA). This test was conducted for three times.

The phospholipid of the ovary, hepatopancreas, and muscle was analyzed using an HPLC system (1200 Series, Agilent, Santa Clara, CA, USA), according to the description of Hang et al., 2015 [29 ]. Cardiolipin, phosphatidylethanolamine, phosphatidylcholine, phosphatidylcholine, and phosphatidylserine (Sigma-Aldrich, St. Louis, MO, USA) were used as standards.
The analysis of carotenoids in each of the three tissues was conducted using a Waters liquid chromatography system (Waters 1525) equipped with a model 2996 photodiode array detector (PAD). The detailed analysis method used is referred to in the report of Bing et al., 2015 [30 ].