Direct detect spectrometer

The top 14 abundant human serum proteins were immunodepleted by High Performance Liquid Chromatography (H PLC) from the pooled serum samples [15] (link). Subsequent to this, samples underwent buffer exchange with 250 mM TEAB (triethlammonium bicarbonate, pH 8.5) and 0.05% SDS (three cycles of buffer exchange) using spin columns (Sartorius Laboratory Products, Victoria, Australia). The protein concentration of the buffer-exchanged sample was determined using a Direct Detect spectrometer (Merck Millipore, Victoria, Australia) and 100 µg of each sample was then reduced with 5 mM TCEP (tris-2 carboxyethyl phosphine) and alkylated with 10 mM MMTS (methyl mehanethiosulfonate).

Cells were grown in a six-well plate, trypsinized, pelleted and washed twice with PBS. The pellet was lysed in lysis buffer (50 mM Tris–HCl, pH 8, 125 mM NaCl, 1% NP-40, 2 mM EDTA, 1 mM PMSF and protease inhibitor cocktail [Roche]) and incubated on ice for 25 min. Samples were centrifuged for 3 min at 12 000 × g and 4°C. Supernatant was collected and protein concentration was determined using the Direct Detect^® Spectrometer (Merck Millipore). Proteins (25 μg) were denatured, reduced, and separated with Bolt® 4–12% Bis–Tris Plus Gel (Thermo Fisher Scientific) in MOPS buffer (Thermo Fisher Scientific, B0001-02). Proteins were then transferred to nitrocellulose membrane and blocked with 5% nonfat milk in TBS-T (50 mM Tris, 150 mM NaCl, 0.1% Tween-20) for 1 h at room temperature. Membranes were incubated with primary antibodies in 5% milk in TBS-T. After overnight incubation at 4°C, the membranes were washed with TBS-T and incubated with HRP secondary antibodies (GE Healthcare Life Sciences, 1:5000), and immunobands were detected with a Pierce ECL Western Blotting Substrate Substrate (Thermo Fischer Scientific, 32106). An uncropped scan of the immunoblot (Figure 2) is shown in Supplementary Figure S20.

An aliquot (2 mL) of untreated and treated samples (bacterial pellet and lyophilizates) with ZEA (described in 2.4.) after 24 h of incubation was centrifuged (RT, 5 min, 14,000 rpm). The obtained pellets were washed with sterile distilled water. Two microliters of samples were dropped on the Assay-free card and allowed to dry [54 (link),55 (link)]. The untreated cells and methanol solution of ZEA served as a blank. The FT-IR spectra were recorded in the range of v = 1350–1850 cm⁻¹ at room temperature using Direct Detect spectrometer (Merck, Darmstadt, Germany).

SW480 (ATCC^® CCL-228™), a cell line established from a primary adenocarcinoma of the colon (Dukes’ type B) and HCT 116 (ATCC^® CCL-247™), a cell line established from a male adult with colorectal carcinoma were obtained from ATCC. NCM460, a cell line derived from normal colon mucosa was obtained from INCELL Corporation LLC (San Antonio, TX, USA) through a licensing agreement. The cell lines were cultured in M3:BaseA^TM (M310A, INCELL Corporation LCC, San Antonio, TX, USA) enriched with 10% fetal bovine serum (FBS) Gibco^TM 10270-098 (Thermo Fisher Scientific, Waltham, MA, USA) to make M3:10A.
Confluent cells were washed three times in PBS-buffer without Ca²⁺ and Mg²⁺ and scraped off. Pellets were further washed with PBS-buffer and kept at −80 °C until use. Cell pellets were re-suspended in SDS sample buffer (LC2676, Invitrogen) for 1D western blotting and in 2D lysis buffer (9M Urea, 2% (vol/vol) Triton X-100, 2% (wt/vol) DTT, 2% (vol/vol) IPG-buffer (GE Healthcare, Buckinhamshire, UK)) for 2D-PAGE analysis and in lysis buffer (5% (wt/vol) SDS, 50 mM TEAB, pH 7.55) for LFQ LC–MS/MS analysis. Protein concentration was determined using the Non-Interfering Protein Assay (488250, Calbiochem^®, Merck KGaA, Darmstadt, Germany) or by infrared spectrometry (Direct Detect Spectrometer, Merck KGaA, Darmstadt, Germany) [48 (link)].

Whole-cell lysates were made from homogenized isolated pulmonary artery samples with radio-immunoprecipitation assay buffer (Abcam, UK) containing a protease inhibitor cocktail (Roche, UK). Protein concentrations were determined through a Direct Detect Spectrometer (Merck, Germany). 20 µg protein from each sample was resolved by SDS-PAGE and transferred to nitrocellulose membranes for Western blotting with antibodies specifically recognizing human AMPK and phosphorylated AMPKα (p-AMPK) (Cell Signaling Technology, US), as well as eNOS, phosphorylated eNOS (p-eNOS), ERA, ERB and GAPDH (Abcam, UK). Immune complexes were visualized using horseradish peroxidase-conjugated secondary antibody with enhanced chemiluminescence on a BioRad Chemidoc MP system.

H2A, H2B, H3, and H4 histones were purified with a commercially available histone purification kit (Active Motif) accordingly to the manufacturer's instruction. Histone concentrations were measured using the Direct Detect^® Spectrometer (EMD Millipore). Heavy and light amino acid-labeled histones were mixed in a 1:1 ratio. Histones were propionylated, quenched by hydroxylamine followed by tryptic digestion overnight and phenyl isocyanate labeling. Histone peptides were then analyzed by capillary reverse phase ultra high-pressure liquid chromatography-electrospray ionization tandem mass spectrometry on an Orbitrap mass spectrometer. Briefly, 1 µg of desalted histone peptides were injected on 1.7 µm BEH-C18 column (Waters) and eluted over the course of 90 minutes with an acetonitrile gradient. Spectra were acquired in a "top-15" data-dependent experiment. Data were further processed with Fishtones (http://research-pub.gene.com/fishtones-js/howto/.)

The samples were sonicated and the protein content was determined by Direct Detect spectrometer (Merck Millipore). Samples were mixed with 6X SDS non-reducing sample buffer (0.35 M Tris, pH 6.8, 10% SDS, 30% glycerol, 0.012% bromophenol blue) and non-boiled samples (8 μg of whole cell lysates) were resolved by 10% SDS-PAGE electrophoresis under non-reducing conditions. Protein samples were transferred onto Immobilon-P PVDF membrane (Merck Millipore) using a semi-dry electroblotter (Biotec-Fischer). Reversible Ponceau S was applied to check equal loading of gels. Immunodetection of NPC1 and beta-actin proteins was performed using a rabbit monoclonal anti-NPC1 antibody (ab134113, Abcam) at a 1:3000 dilution, and a mouse monoclonal anti-beta-actin antibody (mAbcam 8226, Abcam) at a 1:4000 dilution, respectively. Detection was performed by chemiluminiscence using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific). Image capture was carried out using ChemiGeniusQ analysis system and GeneSnap software (Syngene, Cambridge, UK). Images were analysed using GeneTools software package (Syngene).