Advanced protein assay reagent

Advanced Protein Assay Reagent was obtained from Cytoskeleton (ADV01, Denver, CO, USA). Tropomyosin ELISA kit (EL-TPM) was obtained from InBio (Charlottesville, VA, USA).

Before polymerization, actin (20%, pyrene-labeled) in G-buffer was supplemented with 1 mM EGTA and 50 μM MgCl₂ to convert Ca—actin to Mg—actin and then incubated on ice for 10 min [34 (link)]. Polymerization was induced by adding 100 mM KCl, 2 mM MgCl₂, and 0.5 mM ATP. After polymerization for 12 min or overnight at 22°C, 30 μM latrunculin A (Sigma, Tokyo, Japan) was added to induce depolymerization. Rates of polymerization and depolymerization were monitored by measuring the fluorescence intensity of pyrene.
The critical concentrations of WT and G146V actins were measured by sedimentation assays. Different concentrations of actin (0.5 μM~3.0 μM) were polymerized in F-buffer (10 mM HEPES pH 7.4, 2 mM MgCl₂, 100 mM KCl, 0.5 mM ATP, 0.5 mM DTT) for 3 hr at 21°C, and were ultracentrifuged. Amounts of protein in pellets were determined by Advanced Protein Assay Reagent (Cytoskeleton, Denver, CO).

This study was approved by the local ethical committee (Cantonal Institutional Review Board of Basel, Switzerland). In all cases, written informed consent was received. Human term placentas from uncomplicated pregnancies and placentas from cases with preeclampsia were collected in the Department of Obstetrics and Gynecology, University Hospital of Basel, within 1 h following elective or secondary cesarean section. Explants from villous tissue were set in culture in a controlled atmosphere (37°C, 20% oxygen/5% carbon dioxide) as described previously (19 (link)). STBM were isolated from the culture supernatants by a three-step centrifugation procedure at 4°C, namely 1000 × g for 10 min, 10,000 × g for 10 min, and 60,000 × g for 90 min. The microparticle-containing pellet was washed with PBS, re-suspended in PBS/5% sucrose and stored at −20°C until use. The protein content of the STBM was quantified with the Advanced Protein Assay Reagent (Cytoskeleton Inc., Denver, CO, USA) and STBM were standardized for protein concentrations as indicated in the figure legends.

Preparation of the liposomes and protein conjugation were performed as previously described (Ingale et al., 2016 (link)). In brief, liposomes were prepared using a mixture of DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, and DGS-NTA(Ni²⁺) ((1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl) iminodiacetic acid)succinyl] (nitrilotriacetic acid nickel salt)) in a molar ratio of 60:36:4, respectively. The components were dissolved in chloroform, mixed and desicated overnight under vacuum. The formed lipid film was hydrated in PBS for 2 hr at 37°C, with constant shaking followed by sonication. The liposomes were extruded by sequentially passing through 1.0, 0.8, 0.2, and 0.1 μm membrane filters (Whatman Nuclepore Track-Etch membranes). To conjugate the His-tagged trimers, 900 μg total protein was incubated with 300 μl liposomes. The trimer-liposomes were separated from excess free protein using a Superdex 200 size-exclusion column. Trimer-liposome fractions were pooled and quantitated using a standard curve generated by soluble trimer using the Advanced Protein Assay Reagent (Cytoskeleton). Samples were stored at 4°C and checked by EM negative stain analysis and BLI for antigenicity prior to each immunization as previously described (Bale et al., 2017 (link)).

Chitin extracts were prepared by adding chitin samples to PBS-T (100 mg/mL) and vortexing for 30 s. Extraction continued with gentle agitation on a rocking platform for 2 h at room temperature. Samples were centrifuged at 2000× g to allow separation of the protein extract from chitin solids.
APA Buffer was prepared by mixing 4 mL of deionized water and 1 mL of Advanced Protein Assay Reagent (Cytoskeleton Inc., Cat. No. ADV01). For protein quantification, ready APA Buffer (1 mL) was mixed with 10 µL of the chitin extract in an Eppendorf tube and thoroughly mixed by gentle inversion. Similarly, a ‘blank’ was prepared by mixing APA buffer (1 mL) with 10 µL of PBS-T in an Eppendorf tube and mixed by gentle inversion. The samples and ‘blank’ were transferred to a cuvette with 1 cm pathlength. A spectrophotometer (Implen NanoPhotometer™ P-Class, Munich, Germany) was used to measure the OD at 590 nm of the blank sample, which was subtracted from the OD values measured for chitin samples. Protein concentration was determined using the formula OD₅₉₀ = 30 µg protein/mL reagent/cm light pathlength.

The preparation of recombinant dGAE (tau 297–391) has been described previously [30 (link)]. Briefly, dGAE was expressed in bacteria and, following heat treatment, purified by P11 phosphocellulose chromatography. 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.25, was used instead of piperazine-N,N′-bis(ethanesulfonic acid) (PIPES) in some cases. The protein fractions were eluted with 50 mM PIPES (pH 6.8) or 50 mM MES (pH 6.25), each supplemented with 1 mM EGTA, 5 mM ethylenediaminetetraacetic acid (EDTA), 0.2 mM MgCl₂ and 5 mM 2-mercaptoethanol containing 0.1–1 M KCl. The peak for elution of tau, at 0.3–0.5 M KCl, was dialyzed against 80 mM PIPES buffer (pH 6.8), 1 mM EGTA, 5 mM EDTA, 0.2 mM MgCl₂, 5 mM 2-mercaptoethanol, or phosphate buffer (PB) (10 mM; pH 7.4). The dGAE protein concentration. was measured using Advanced Protein Assay Reagent (Cytoskeleton, Inc. Denver, CO, USA.) using bovine serum albumin as a standard. The protein was diluted with 10 mM phosphate buffer (pH 7.4) and, for all experiments, the dGAE was used at a concentration of 100 μM (Table 1).