Bradford colorimetric assay

Six different snap-frozen blocks of frontal cortex from 3 independent SPMS cases and one block from 6 different control cases were processed for protein extraction. Detergent-soluble proteins were extracted with Ripa buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS in PBS, containing protease inhibitors), using a micropestle. After a short sonication, the homogenates were incubated on ice for 1 h and centrifuged at 12,000 rpm for 10 min at 4 °C. Protein quantification was performed from the supernatants by Bradford colorimetric assay (Biorad, Milan, Italy). Proteins (30 μg) were separated by gel electrophoresis on 10% SDS-PAGE and transferred to nitrocellulose Hybond-C-extra membranes (Amersham Biosciences, Buckinghamshire, UK). Filters were pre-wetted in 5% blocking agent in TBS-T (10 mM Tris pH 8, 150 mM NaCl, 0.1% Tween 20) and hybridized overnight with H1 receptor polyclonal antiserum (1:500, Santa Cruz) or HNMT polyclonal antiserum (1:400, Atlas Antibodies). Signals were detected with anti-rabbit horseradish peroxidase-conjugated antibody (1:5000) and developed by enhanced chemiluminescence (Amersham Biosciences) using Kodak Image Station (KDS IS440CF). Semi-quantitative analysis was performed with Image J software.

Approximately 5 × 10⁵ DU145 cells were resuspended in hypotonic lysis buffer (10 mM HEPES, 50 mM NaCl, 2 mM EDTA, 5 mM DTT, 0.1% CHAPS, 1 mM PMSF, and 10% Sucrose, pH 7.4) and stored at –80 °C. The lysate was then subjected to four freeze-thaw cycles before centrifugation at 10,000 g for 10 min. Protein concentrations were determined in the supernatants by the Bradford colorimetric assay (Biorad, USA). The reaction was started at 37 °C by incubating 100 μg of total protein with caspase-specific substrates (caspase-3, DEVD-AFC; caspase-8, IETD-AFC; caspase-9: LEHD-AFC) in assay buffer (0.05 M HEPES, pH 7.4, 10 mM DTT, 100 mM NaCl, 1 mM EDTA, 0.1% CHAPS, 10% sucrose) and allowed to incubate at 37 °C for 45 min. Protease activity was monitored every 30 s within a total period of 30 min at an excitation wavelength of 400 nm and an emission wavelength of 510 nm using a SpectraMax Gemini XPS spectrofluorometer (Molecular Devices, CA, USA). Caspase activity was calculated per microgram of protein, and expressed as a percentage of control activity. Assays were performed in the presence or the absence of the pan-caspase inhibitor 1 μM ZVAD-FMK as control for broad specificity of caspase activity.

MDA-MB-231 cells grown in six-well plates at 37 °C and treated with the tribodies at a concentration of 200 nM for 72 h were collected and lysed, as previously reported [44 (link)]. Protein concentrations were determined by Bradford colorimetric assay (Biorad, California, USA), and Western blotting analyses were performed by incubating the membranes with the anti-phospho-p44/42 MAPK, anti-p44/42 MAPK, anti-phospho-Sapk/Jnk, anti-Sapk/Jnk, anti-cleaved Caspase-3, anti-Caspase 3 and anti-vinculin antibodies (Cell Signaling Technology, Danvers, MA, USA), followed by incubation with the appropriate HRP-conjugated secondary antibodies and detection, as previously reported [45 (link)].

Recombinant NikR protein was overexpressed and purified under native conditions as previously described19 (link). The purified protein was dialyzed overnight against NikR footprinting buffer (20 mM Hepes pH 7.85, 50 mM KCl, 0.02% Igepal, 0.1 mM DTT and 10% glycerol) prior to the DNA binding experiments. A Bradford colorimetric assay (Bio–Rad) was used to quantify the protein fractions with bovine serum albumin as standard.

To quantify rTvSaplip12 content in the soluble fraction, after total soluble protein quantification using a Bradford colorimetric assay (Bio-Rad, Hercules, CA), Enzyme Linked ImmunoSorbent Assay Maxisorp 96-well microtiter plates (NUNC) were coated in triplicate with 50 μg of proteins per well. After washing and blocking, the plates were incubated with the anti-FLAG M2 HRP-conjugated (A-8592, Sigma Chemical Co., MO) antibody, diluted 1:5,000 in 2% milk (w/v) in 1× PBS. Bound antibodies were revealed using 2,2′-azino-di-3-ethylbenz-thiazoline sulphonate (ABTS; KPL Inc., Gaithersburg, MD), and the colorimetric reaction measured with an ELISA reader at 405 nm. For the calibration curve a quantified purified protein fused to FLAG tag has been used.

Synaptosomes were individually prepared from 6 WT and 6 KO mice, as well as 6 WT mice treated with CTEP or vehicle as previously described [37 (link)]. Hippocampi were homogenized in HEPES buffer (5 mM HEPES, pH 7.4, 0.32 M sucrose, with EDTA-free protease inhibitor cocktail, Roche, Basel, Switzerland) in a dounce homogenizer (12 strokes, 900 rpm). The homogenate was centrifuged at 1000× g for 10 min and the supernatant was subsequently centrifuged in a 0.85/1.2M sucrose gradient at 100,000× g for 2 h. The synaptosomes collected from the 0.85/1.2 M sucrose interface were concentrated by centrifugation at 18,000× g for 20 min. Protein concentration was determined by using Bradford colorimetric assay (Bio-Rad, Hercules, CA, USA; 5000006EDU).

The ubiquitination status of NLRP3 protein was detected using immunoprecipitation. BMDMs were treated with 20 µM of MG132 (Selleckchem, Houston, TX, USA) before LPS and Nigericin stimulations. The pellets were collected and lysed with T-PER Tissue Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with proteinase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). The total protein concentration was established using the Bradford colorimetric assay (Bio-Rad Laboratories, Richmond, CA, USA). Endogenous NLRP3 was immunoprecipitated with an antibody for NLRP3 (#15101, Cell Signaling, Technology, Danvers, MA, USA, 1:50) and Dynabeads Protein A (Thermo Fisher Scientific, Waltham, MA, USA) at room temperature. For each sample, an equal protein concentration was added to the antibody-coated beads and incubated at room temperature. After that, the beads were washed twice with washing buffer, resuspended in Laemmli Sample Buffer 2X (Biorad Laboratories, Hercules, CA, USA), and boiled at 95 °C for 5 min. Immunoprecipitated samples were analyzed by immunoblotting.