Pageruler prestained

Antibody binding was verified by western blot on fractionated mouse brain tissue as previously described [9 (link), 15 (link)]. Protein amount was 100μg per well and the protein transfer was performed with the Trans-Blot® Turbo™ Mini PVDF Transfer Packs and Trans-blot Turbo Transfer system (Bio-Rad), following the manufacturer’s instructions. Anti-MFSD4A (1:100, rabbit, AV53395, Sigma-Aldrich) and anti-MFSD9 (1:50, goat, sc-247973, Santa Cruz) were used as antibodies, and a molecular weight marker (PageRuler Prestained, Thermo Fisher Scientific) was included as reference on each blot. HRP-coupled secondary antibodies (anti-rabbit and anti-goat (Invitrogen) dilution 1:10000) were added followed by chemiluminescent development using Clarity Western ECL Substrate (Bio-Rad). Staining was visualized using a CCD camera (Bio-Rad). Glycosylation sites for the mouse proteins were predicted using the NetOGlyc 4.0 Server from CBS Predictions Servers [48 (link)].

Zymographic analysis of the culture supernatant of the selected bacterial isolate was performed. The sample was mixed at 1:1 ratio with the sample buffer (Tris–HCl 0.32 M; pH 6.8; glycerol 48%; SDS 8%; bromophenol blue 0.06%). Sample in the amount of 5 or 10 μL was loaded onto 12% polyacrylamide gel (5% staking gel) containing 0.1% of copolymerized casein. PAGE Ruler prestained (Thermo Scientific) was used as a reference marker. Electrophoresis was performed at constant 18 mA, at 2 °C. Subsequently, the gel was washed twice with Triton-X 2.5%, once with the incubation buffer and incubated for 24 h at 28 °C in the same buffer (Tris–HCl 0.05 M, pH 7.5, containing CaCl₂ 2 mM and NaN₃ 0.02%). Proteolytic activity bands were visualized by staining with Coomassie Blue and decolorization with methanol: acetic acid: water (50:10:40).

Protein isolation from cultured fibroblasts of individual III-1 from family A and Western blot were carried out as described [6 (link)]. The primary antibodies used for immunodetection were rabbit anti-WDR11 (Novus Biologicals, NBP1-89930, 1:500, N-terminal epitope aa 268-348) and rabbit anti-WDR11 (Abcam, ab93871, 1:500, C-terminal epitope aa 1174–1224). Rabbit anti-α-Tubulin (Abcam, ab15246) and mouse anti-beta-Actin (Abcam, ab6276) antibodies were used to control equal loading of protein extracts. As secondary antibodies, horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgGs (Santa Cruz Biotechnology, sc-2370/sc-2005) were used. PageRuler Prestained (ThermoScientific) and biotinylated Protein Ladders (Cell Signaling, 7727S) were used for protein molecular weight estimation. Detection was done using Clarity Western ECL Substrate (Bio-Rad) and the FujiFilm LAS 3000 system.

The chemicals used were obtained from the following sources: arachidonic acid (5Z,8Z,11Z,14Z-eicosatetraenoic acid) and linoleic acids from Cayman Chem (distributed by Biomol, Hamburg, Germany), HPLC grade methanol, acetonitril and acetic acid from AplyChem (Darmstadt, Germany), isopropyl-β-d-thiogalactopyranoside (IPTG) from Molecula (Munich, Germany), and L-a-Phosphatidylcholine from Avanti Polar Lipids (Alabaster, AL, USA). The E. coli strain BL21(DE3)pLysS were purchased from Invitrogen (Carlsbad, CA, USA). Peptone medium was obtained from Greenvan (Moscow, Russia), glucose from Biotech Rosva (Kaluga, Russia), antifoam «Sofexil-1520» from Sofex silicone (Moscow, Russia), ampicillin from ApliChem (Darmstadt, Germany) and thiamine hydrochloride from Sigma-Aldrich (Darmstadt, Germany). Western-blotting was performed with anti-His-tag antibodies-HRP from Sigma-Aldrich (Darmstadt, Germany). Protein mass marker PageRuler™ Prestained was purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Dried protein pellets were dissolved in 2D sample buffer (in mM): 7000 urea, 2000 thiourea, 4% Chaps, 1% TritonX-100, 32.4 DTT, carrier ampholytes (Bio-Lyte® 3/10 Ampholyte Biorad #163-2094); at 37 °C. The PROTEAN® IEF System from Biorad and 17 cm IPG strips pH 4-7 (IPG ReadyStrip™ Biorad #163-2008) were used for isoelectric focusing (IEF). The strips were allowed to rehydrate overnight and focused as described (PROTEAN® IEF Cell Instruction Manual). After IEF each strip was equilibrated in DTT and IAA equilibration buffer (in mM): 50 Tris pH 8.8, 6000 urea, 33% glycerol, 4% SDS, EB1: 130 DTT, EB2: 162 IAA; for 20 min. Half of each strip (pH 4-5.3, ca. 7.5 cm) was opposed to the second dimension gel (hand-cast 12% polyacrylamide gel, Mini-PROTEAN® Cell Biorad) and run for 1 h at 7 mA per gel and 3.5 h at 15 mA per gel in Tris-Glycine buffer. A molecular weight marker (PageRuler™ prestained, ThermoFisher #26617) was moved into a small piece of filter paper and placed in front of the IPG strip before the gel run. For some validation experiments an additional marker was placed at the end of the strip (Precision Plus Protein^TM WesternC^TM Blotting Standards, Biorad #1610385). 2D separated gels were either used for protein transfer to PVDF membranes and immunostaining or high-sensitive silver staining followed by cutting of protein-spots and mass spectrometry analysis.