Mini protean apparatus

The ELISA kit to measure corticosterone was from Enzo Life Science. Rabbit polyclonal antibodies anti-TREK-1 and anti-proBDNF were from Alomone Labs (Israel). Rabbit polyclonal antibodies anti-furin and anti-NaKATPase, the mouse monoclonal anti-beta-actin and goat anti-TGN38 and anti-DCX antibodies were from SantaCruz Technologies (United States). Rabbit polyclonal antibodies anti-TrkB and anti-phospho-(Tyr 705)-TrkB were from Signalway Antibody (United States). Rabbit polyclonal antibodies against BDNF and plasminogen were from GeneTex (United States). Mouse monoclonal anti-BrdU antibody was from Becton Dickinson (United States). Anti-rabbit, anti-mouse, and anti-goat secondary antibodies were from Cell Signaling Technologies. HRP substrate (Immobilon Forte, Millipore), Nitrocellulose (BioTrace NT, Life Sciences), SDS-PAGE gels and Mini Protean apparatus were from Biorad. BrdU was from Interchim (France). Neurobasal medium, B-27 and Penicillin-Streptomycin were from Thermo Fisher Scientific. Poly-D-Lysine was from Sigma-Aldrich. Patch clamp was performed on a pClamp 8.2 apparatus (Molecular Devices)

A 194-bp DNA fragment harboring the promoter region of the dinJ-yafQ operon was PCR-amplified under standard conditions from L. paracasei 4366 total DNA by using primers labeled with DY682 fluorophore (Table S2). The EMSA reactions (total volume 10 μL) containing 2 nM DNA, 128 ng of salmon sperm DNA, and increasing concentrations of DinJ or DinJ-YafQ complex in buffer 50 mM Tris-HCl, pH 7.5, 50 mM KCl, 5% glycerol were prepared at room temperature and incubated for 10 min. The samples were electrophoresed on a 6% native polyacrylamide gel in TBE buffer (45 mM Tris, 45 mM boric acid, 1 mM EDTA, pH 8.3) for 70 min at 90 V at room temperature using a Mini-Protean apparatus (Bio-Rad). The gel image was recorded using a ChemiDoc MP imager and unbound DNA bands were quantified by densitometric analysis using Image Lab software (Bio-Rad). The fraction of bound DNA, determined with respect to the lane without protein added, was plotted as a function of protein concentrations and fitted by using a Hill equation: $\mathrm{fraction}\mathrm{of}\mathrm{bound}\mathrm{DNA}=1/(1+{(K_{\mathrm{Dapp}}/\left[P\right])}^n)$ , where K_Dapp is the apparent dissociation constant, [P] the DinJ or DinJ-YafQ protein concentration, and n the Hill constant.

Whole-cell extracts were prepared by washing the cells in cold PBS, pH 7.2, followed by solubilizing them in hot SDS-PAGE sample buffer (35 (link)), sonicating three times for 5 s at 20^oC, and boiling for 5 min. Alternatively, in experiments analyzing sphingolipids or studies aimed to quantify expression of selected proteins (load), 2× concentrated SDS-PAGE sample buffer was added to the aliquots of sonicated lysates. Protein extracts (25 μg) were size fractionated in 13% SDS-PAGE gels and electrophoretically transferred onto nitrocellulose membrane (0.45 μm; GE Healthcare Life Sciences) using a Bio-Rad Mini-PROTEAN apparatus. The membranes were blocked with 2% BSA-TNT solution (20 mM Tris-HCl, pH 7.5, 200 mM NaCl, and 0.1% Tween) and analyzed by immunoblotting with protein-specific antibodies. Bound primary antibodies were detected with the corresponding HRP-conjugated secondary antibodies. HRP signal in loads and phosphorylation studies was detected with chemiluminescence reagent (36 (link)), and HRP signals in pull-down studies were developed with WesternBright ECL HRP substrate (Advansta) or SuperSignal™ West Femto (Thermo Fisher Scientific) and collected with Luminescent Image Analyzer LAS 3000 (FujiFilm). Aida Image Analyzer, version 5.0 software (Raytest) was used for signal quantification.

Collagen molecular weight (MW) was determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to the Laemmli method [42 (link)]. Briefly, the lyophilized collagen was dissolved in 0.1 M acetic acid and mixed at a 1:2 (v/v) ratio with sample buffer (0.5 M Tris-HCl, pH 6.8; containing 5% SDS, 20% glycerol, 5% β-ME, and 0.2% bromophenol blue). The mixed solution was incubated at 95 °C for 5 min. A 10 μL sample was processed using discontinuous polyacrylamide gel electrophoresis (7.5% separator and 4% stacking). A molecular weight protein marker was used to estimate collagen MW. The electrophoresis analysis was run at a 25 mA constant current voltage using a Mini-Protean apparatus (Bio-Rad Laboratories, Watford, UK). Protein bands were stained using Coomassie Brilliant Blue R250 solution.

All chemicals and reagents were of analytical grade and from commercial sources. Acetonitrile (ACN), Tris(hydroxymethyl)aminomethane (Tris–HCl), hydrochloric acid (HCl), ammonium bicarbonate, 2-iodoacetamide (IAM), 1,4-dithiothreitol (DTT), and trypsin from bovine pancreas (T1426, lyophilized powder, ≥10,000 units/mg protein) were from Sigma-Aldrich (St. Louis, MO, USA). Bovine serum albumin (BSA), Mini-Protean apparatus, precision plus protein standards, Bradford reagent, and Coomassie Blue G-250 were purchased from Bio-Rad (Hercules, CA, USA). The liquid bacterial α-amylase used for starch hydrolysis and the liquid preparation Cellustar XL (containing cellulase and xylanase) used for the hydrolysis of non-starch polysaccharides were obtained from AB Baltic Enzymes (Vilnius, Lithuania), whereas the neutral protease SQzyme PS-NL used for protein hydrolysis was from SUNTAQ (Guangzhou, China).

Proteins from individual saliva samples of all animals (both without and with acid stimulation) were separated by SDS-PAGE electrophoresis in 14% acrylamide gels in a mini-protean apparatus (BioRad) as described before [23 (link)]. Briefly, a total of 15 μg protein from each saliva sample was run in each lane. The samples were resuspended in sample buffer [Tris–HCl 0.125 M pH 6.8, 2% (w/v) SDS, 5% (v/v) 2-mercaptoetanol, 20% (v/v) glycerol traces of bromophenol blue], heated at 95°C for 5 minutes, and run at a constant voltage of 140 V until the dye front reaches the end of the gel. Gels were fixed in 40% methanol, 20% acetic acid, for one hour, stained with Coomassie Brilliant Blue (CBB) G-250 (0.125% CBB G-250, 20% ethanol) for two hours and destained in several washes with distilled water. A scanning Molecular Dynamics densitometer with internal calibration and LabScan software (GE, Healthcare) were used to acquire gel images and to determine the percentage of volume of each protein band; GelAnalyzer software (http://www.gelanalyzer.com/) was used to analyze the gel images. Molecular masses were determined in accordance with molecular mass standards (Bio-Rad Precision Plus Protein Dual Color 161–0394) run with protein samples.

The immunogenicity of the P. orientalis salivary proteins was tested by the immunoblot technique. Salivary gland homogenate (4.2 μg of total salivary proteins per lane, equivalent to 30 glands) and recombinant proteins mAG5, mYEL1, and mAG5 + mYEL1 (3 μg of each protein per lane) were electrophoretically separated on a 12% polyacrylamide gel under non-reducing conditions using a Mini-protean apparatus (Bio-Rad). Separated protein bands were transferred onto a nitrocellulose membrane using the iBLOT system (Invitrogen) and then cut into strips. Strips were either stained with amidoblack (Merck; 0.1% solution in 25% isopropanol and 10% acetic acid) or blocked in 5% non-fat milk diluted in Tris-buffered saline with 0.05% Tween 20 (TBS-Tw) overnight at 4°C and subsequently incubated for 1 hour with human sera diluted 1:50 in TBS-Tw. After the washing step with TBS-Tw, the strips were incubated with peroxidase-conjugated Anti-Human IgG Antibody (Sigma-Aldrich) diluted 1:500 in TBS-Tw. The chromogenic reaction was developed using a substrate solution containing diaminobenzidine and H₂O₂.