Pageruler prestained protein ladder

The SOD forms were separated by native electrophoresis on 12% (w:v) polyacrylamide gels at a constant voltage of 180 V for about 1 h [64 (link)]. A total of 30 µg of protein was loaded onto each lane. The gels were stained for SOD activity according to the method described by Beauchamp and Fridovich [65 (link)]. After electrophoresis and incubation for 20–25 min in the dark at room temperature in the standard staining buffer: 50 mM phosphate buffer, pH 7.8, 10 mM EDTA, 28 mM TEMED, 30 µM riboflavin and 245 µM nitro blue tetrazolium chloride (NBT), the gels were exposed to the artificial white light until the SOD activity bands became visible. To inhibit Cu/ZnSOD and FeSOD, the gels were stained in a standard buffer containing 5 mM H₂O₂. Inhibition of Cu/ZnSOD was achieved in the presence of 2 mM KCN. In addition, a protein mass ladder (PageRuler™ Prestained Protein Ladder, Thermo Scientific, Waltham, MA, USA) was used to estimate the molecular weight (MW) of the separated SOD forms (see also Supplementary Material Figure S1, Tables S1 and S2).

TSPO protein levels of were analyzed under non-reducing conditions. For this, tissue samples and primary microglia were lysed by sonication in ice cold PBS supplemented with protease and phosphatase inhibitors (Complete protease inhibitor cocktail, Roche). Protein concentration was determined by BCA Protein Assay according to the manufacturer’s instructions (Thermo Scientific). Equal amounts of samples were heated in SDS sample buffer containing only 0.2% SDS and no β-mercaptoethanol and then loaded onto 12% tris–glycine polyacrylamide gels and run under standard conditions. For immunoblotting, proteins were electrophoretically transferred onto a 0.45 µm nitrocellulose membrane (Bio Rad) at 100 V for 1 h. Membranes were blocked with 5% nonfat dried milk powder in TBS‐T before incubation with primary antibody against TSPO (1:1000 dilution, rabbit polyclonal, ab109497, Abcam) or Actin (1:1000 dilution, chicken anti‐b‐Actin clone AC‐15, A5441, Sigma‐Aldrich). After several washing steps in TBS-T, membranes were incubated with horseradish peroxidase‐conjugated secondary antibodies (1:4000 dilution, Dako) and the immune complex was visualized by a MultiImage II system (Alpha Innotech). PageRuler pre-stained protein ladder (Thermo Scientific) was used for identification of protein size. Band intensities were quantified using ImageJ.

Protein samples were incubated at 70°C for 10 min after adding 3× SDS sample buffer (stock concentration 30% glycerol, 3% SDS, 93.75 mM Tris‐Cl pH 6.8, 0.06% bromophenol blue). These samples were loaded on SDS–PAGE gels (8 or 12%) and run at 90 V. After dye front reached the end, these gels were transferred with TransBlot (Biorad) at conditions of 1.0 mA for 30 min onto PVDF membranes. Transferred membranes were blocked with 5% skim milk in TBST, and antibodies were added subsequently and incubated overnight at 4°C. The following antibodies were used; Flag‐HRP (Sigma, A8592), Myc‐HRP (Sigma, 16‐213), V5‐HRP (Sigma, V2260), HA‐HRP (Roche, 12013819001), MPK6 (Agrisera, AS12 2633), H⁺‐ATPase (Agrisera, AS07 260), and GFP‐HRP (Abcam, ab6663). Signals were detected using ECL substrates (Thermo Fisher, 34580). After detection, membranes were stained with Ponceau S solution (Sigma, P7170) to use as loading control. PageRuler™ Prestained Protein Ladder (Thermo Scientific, 26616) was used as molecular weight markers.

For SDS-PAGE electrophoresis, phages ϕPD10.3 and ϕPD23.1 (ca. 10¹⁰ pfu ml^-1) were diluted two times with sterile demineralized water. Laemmli buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue and 0.125 M Tris-HCl, pH 6.8, 6 × concentrated stock) was added to the samples to obtain 2 × concentrated working solution and the mixture was firstly frozen in liquid nitrogen for 1–2 min and then boiled for 5 min at 95°C. Twenty five μl of phage extracts were separated in 12% acrylamide SDS-PAGE gel (Rothiphorese Gel 30, 37.5:1) (ROTH) for 19 h at 50 V at room temperature (22°C). PageRuler Pre-stained Protein Ladder (Thermo Scientific), prepared according to instructions provided by the manufacturer, was used as a size marker. The gel was stained with PageBlue Coomasie Blue (Thermo Scientific) for 18 h and destained with sterile demineralized water for 6 h at room temperature (22°C) as suggested by the manufacturer. Protein bands were excised from the gel with a sterile scalpel and used for mass spectrometry analysis.

The protein lysates were separated in the presence of SDS (Roth, Karlsruhe, Germany) using 10% polyacrylamide gel, according to the Laemmli method [38 (link)] and visualized with Coomassie Brilliant Blue R-250 (Roth, Karlsruhe, Germany) or transferred to the nitrocellulose membrane (Roth, Karlsruhe, Germany). The PageRuler Prestained Protein Ladder (Thermo Fisher Scientific, Waltham, MA, USA) was used as a protein standard. The proteins fractionated by SDS-PAGE were transferred to nitrocellulose membrane [39 (link)], overlaid with anti-P1 antibodies (diluted 1:100) or Stx1B (final concentration 1 µg/mL and anti-6xHis antibody diluted 1:1000 as secondary) and anti-mouse antibody conjugated with alkaline phosphatase (diluted 1:1000) for 45 min in room temperature and visualized using NBT/BCIP reagents (nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate) (Roth, Karlsruhe, Germany) in 100 mM Tris pH 9.2 with the addition of 1 mM MgCl₂, 1 mM MnCl₂ and 150 mM NaCl.

Protein contents of crude preparations or partially purified fractions were determined by the Bradford dye-binding method using a pre-fabricated assay (Biorad Laboratories, Marnes-la-Coquette, France) and BSA as calibration standard. Protein concentrations of purified enzymes were measured based on their molar absorption coefficients calculated from the mature amino acid sequence using ProtParam (http://www.web.expasy.org/protparam/). The molar absorption coefficients of PsLPMOA and PsLPMOB at 280 nm are 30.62 and 33.14 mM⁻¹ cm⁻¹, respectively. Concentrations of NcCDHIIA and NcCDHIIB were determined at 420 nm (103 and 87 mM⁻¹ cm⁻¹, respectively). Protein purification steps and fermentation progress were tracked by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad Laboratories). Protein bands were visualized with Coomassie Brilliant Blue R-250. The molecular mass under denaturing conditions was determined with reference standard proteins (PageRuler Prestained Protein Ladder, Thermo Fisher Scientific). All procedures followed the manufacturer’s recommendations (Bio-Rad Laboratories). Electrophoresed proteins were electroblotted onto polyvinylidene difluoride membranes following the manufacturer’s procedure (iBlot, Life Technologies, Saint-Aubin, France) [40 (link)].