Tropix 1 block

Following 2D electrophoresis, gels were washed sequentially in depurination buffer (0.125 M HCl), denaturation buffer (0.5 M NaOH, 1.5 M NaCl) and neutralisation buffer (0.5 M Tris–HCl, 1.5 M NaCl pH 7.5) with washes in ddH₂O in-between each buffer. DNA was transferred onto Hybond-N+ membrane (GE Healthcare) by capillary action in 20× SCC (3 M NaCl, 350 mM NaOC trisodium citrate pH 7.0). Membranes were cross-linked using a UV Stratalinker 1800 (Stratagene) at 1200 J/m and subsequently blocked in hybridisation buffer (5× SSC, 5% Dextran sulphate (Sigma-Aldrich, D8906) 0.2% Tropix I-Block (Applied Biosystems, T2015), 0.1% SDS) for at least 1 h at 60°C.
Catenated pRS316 plasmids or replication intermediates from pRS426-RFB were probed with DNA amplified from pRS316 (probing specifically for the URA3 gene). Labelling and detection used random prime labelling incorporating fluorescein tagged dUTP (Roche). Following probing, hybridized fluorescein tagged dUTP was detected with alkaline phosphatase tagged anti fluorescein Fab fragments (Roche), revealed with CDP-Star (GE Healthcare) and non-saturating exposures acquired on an ImageQuant LAS4000 system (GE Healthcare). Densitometry analysis was carried out using ImageQuant TL software.

VSMCs were washed in ice-cold PBS and solubilized with 4% sodium dodecyl sulfate. Femoral arteries were homogenized with lysis buffer (20 mM Tris-HCl, pH 7.4, containing 1% Nonidet P-40, 150 mM NaCl, and protease inhibitor cocktail). The homogenates were centrifuged at 13,000 ×g for 15 min, and the resulting supernatants were used for immunoblot analysis. Protein concentration was determined using Pierce BCA Protein Assay Kit (Thermo Scientific) according to the manufacturer’s instructions. Protein samples obtained from vessels or cells were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Membranes were blocked with TBS containing 0.2% Tropix I-block (Applied Biosystems) and 0.2% Tween 20 and were incubated overnight at 4 °C with primary antibodies. After incubating with secondary antibodies, proteins were detected by ECL (GE Healthcare, Little Chalfont, United Kingdom) chemiluminescence. Molecular weight was calculated with pre-stained protein marker that was applied to the same gel run samples. The relative densities were analyzed using ImageQuant TL software (GE Healthcare).

Western blot analysis was performed to detect antibodies to relapsing fever spirochetes using a whole-cell lysate of B. hermsii DAH and a purified recombinant GlpQ protein from the same strain [18 (link)]. The protein preparations were separated by electrophoresis in Novex® 4–20 % Glycine Gels 1.0 mm (Invitrogen, Life Technologies, Grand Island, NY, USA), and transferred to nitrocellulose membranes using the iBlot® Gel Transfer Device according to the manufacturer’s instructions (Invitrogen, Life Technologies). Membranes were blocked in Tropix® I-BLOCK (Applied Biosystems, Life Technologies, Grand Island, NY, USA) at room temperature for 1 h. Serum samples were diluted 1:100 in 5 ml of I-BLOCK and incubated with the membrane at room temperature for 1 h. Membranes were removed from the serum samples, washed with I-BLOCK, and incubated with HRP-conjugated recombinant Protein A (1:4,000) (Invitrogen, Life Technologies). The membranes were washed in I-BLOCK for 2 h with four changes of the wash solution, and then developed for ~30 s using ECL® Western Blotting Detection Reagents (GE Healthcare, Little Chalfont, UK). Positive and negative control samples were obtained from infected and uninfected laboratory mice. A serum sample was considered positive if it contained antibodies that bound to eight or more proteins in the B. hermsii whole-cell lysate and to the purified GlpQ.

Blocking of nonspecific interactions was tested by bovine serum albumin (BSA) (Sigma-Aldrich Chemie GmbH, Darmstadt, Germany, catalog number: A8806), I-block (Tropix I-block, Applied Biosystems, Waltman, MA, USA, catalog number: T2015), Poly-(L-lysine)-graft-poly(ethylene-glycol) (PLL-g-PEG, SuSoS AG, Dübendorf, Switzerland, shortened as PP later on), poly(acryl-amide)-g-(PMOXA, 1,6-hexanediamine, 3-aminopropyldimethylethoxysilane) (PAcrAM-graft-(PMOXA, NH₂, Si), SuSoS AG, Dübendorf, Switzerland, shortened as Pacram-P thereafter), which were dissolved in MES buffer, PBS (Sigma-Aldrich Chemie GmbH, Darmstadt, Germany, catalog number: P4417), 10 mM and 1 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) buffer, respectively.