Softmax pro software v5

Proteins from tomato leaves of four biological replicates were prepared according to Hurkman and Tanaka50 (link) with the following modifications. Samples were ground in liquid nitrogen into fine powder and incubated in extraction buffer (0.1 M Tris-HCl pH 8.8, 10 mM EDTA, 0.2 M DL-Dithiothreitol, 0.9 M sucrose), continued by grinding in a fume hood, and then the extract was agitated for 2hrs at room temp. After washing twice with 0.1 M ammonium acetate in methanol and twice with 80% acetone, the dried pellet was dissolved with 2D buffer (8 M Urea, 4% CHAPS, 40 mM Tris-base, 2 M Thiourea). Protein assays were performed using an EZQ Protein Quantitation Kit (Invitrogen, Carlsbad, CA, USA) with the SoftMax Pro Software v5.3 (Molecular Devices, Downingtown, PA, USA).

Biological pentaplicates (i.e. n = 5 replicates per S. mutans strain per growth condition, including the n = 3 replicates analyzed previously by RNA-seq.^{7 (link)}) of each S. mutans culture were processed for protein extraction according to Fujiki et al.^{18 (link)} with the following modifications: Samples were ground into fine powder in liquid nitrogen and agitated in extraction buffer (0.1 M Tris-HCl pH 8.8, 10 mM EDTA, 0.2 M DTT, 0.9 M sucrose) with an equal volume of phenol (pH 8.0) for 2 hours at room temperature. The phenol phase was precipitated by adding five volumes of 0.1 M ammonium acetate in methanol. After washing twice with 0.1 M ammonium acetate in methanol and twice with 80% acetone, the dried pellet was dissolved with 50 mM ammonium bicarbonate buffer. The mixtures were incubated on ice before centrifugation at 4 °C for 20 minutes at 12,000 × g, separating the proteins into soluble and insoluble phases. The insoluble phase was incubated with protein buffer (8 M urea, 2 M thiourea, 2% ASB-14 (amidosulfobetaine 14)) for at least one hour with occasional vortexing. The proteins were quantified using an EZQ Protein Quantitation Kit (Invitrogen, Carlsbad, CA, USA) and SoftMax Pro Software v5.3 (Molecular Devices, Downingtown, PA, USA).

Coral fragments that were incubated for 48 h were subjected to proteomic analysis. Previously frozen samples were thawed and tissue was removed from the calcium carbonate skeleton over ice using a Paansche airbrush (Paansche, Inc., Chicago, IL, USA) and homogenate buffer (50 mM phosphate buffer, (pH 7.8) with 0.05 mM dithiothreitol). Four biological replicates were used per treatment (total = 20). Total protein was isolated from crude tissue homogenates (~2 g of coral tissue) as described previously [61 (link)] with the following modifications. Tissue was ground in liquid N₂ with a pre-cooled mortar and pestle in 6 mL of extraction buffer (1.2% β-mercaptoethanol, 0.1 M Tris-HCl (pH 8.8), 10 mM EDTA, 0.9 M sucrose) and 6 mL Tris-saturated phenol (pH 8.8), followed by overnight incubation at room temperature with shaking. All chemicals were obtained from Millipore Sigma (St. Louis, MO, USA) unless noted otherwise. Samples were centrifuged at room temperature for 40 min at 5,000 g. The resulting protein pellets were dissolved in 4 M urea and 0.1% SDS in 10 mM Tris-HCl (pH 8.0). Protein concentration was measured using the EZQ Protein Quantification Kit (Thermo Fisher Scientific, Inc., San Jose, CA, USA) with SoftMax Pro Software v5.3 (Molecular Devices, Inc., San Jose, CA, USA).