1 4 dithiothreitol dtt

Extracts were dried (Concentrator Plus; Eppendorf, Hamburg, Germany) for 2 h and reconstituted in 100 µl of ammonium bicarbonate (ABC; 50 mM) with the aid of sonication (Branson 1-291; Branson, Danbury, CT) for 10 min. To reduce disulfide bonds, 2.4 µl of DTT (1,4-dithiothreitol; Sigma-Aldrich) (10 mM) was added followed by incubation at 60°C for 45 min. Alkylation was performed using 5 µl of 30 mM iodoacetamide (Sigma-Aldrich) and incubation at room temperature in the dark for 45 min. Subsequently, 10 µl of trypsin (Sigma-Aldrich) diluted in ammonium bicarbonate (ABC) (100 ng/µl) was added. Samples were maintained at 37°C overnight, and then 2 µl of formic acid (Optima; Fisher Chemical, Wilmington, DE, USA) (liquid chromatography/mass spectrometry [LC/MS] grade) was added, followed by incubation of the samples at 37°C for 10 min. After centrifugation (1,400 rpm, 5 min), the supernatant was transferred to another tube and dried for 2 h (Concentrator Plus; Eppendorf, Hamburg, Germany). A 200-µl volume of 0.1% formic acid (Optima, Solvent Blends; Fisher Chemical) (LC/MS grade)–water was used to reconstitute the samples for analysis. As a control, HeLa Protein Digest Standard (Pierce–Thermo-Fisher) was used.

A sample of OFM+Mϕ was resuspended in PBS to a final protein concentration of 0.1 mg/mL. Samples (20 μg) were reduced with 10 mM 1,4-dithiothreitol (DTT) (Sigma-Aldrich) (20 μL, 60 mins, 60°C), then alkylated with 20 mM iodoacetamide (Sigma-Aldrich) (20 μL, 30 mins, rt°C in the dark). Sample (60 μL) were digested overnight (37°C) with 0.1 μg trypsin (Sigma-Aldrich). The sample was dried, then reconstituted in loading buffer (0.1 M sodium bicarbonate) prior to ESI MS/MS analysis.

Roswell Park Memorial Institute (RPMI)-1640 (Invitrogen, Carlsbad, CA, USA), fetal bovine serum (FBS) (Invitrogen), 1% antibiotic-antimycotic (AA) solution (Invitrogen), Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Invitrogen), 1,4-dithiothreitol (DTT) (Sigma-Aldrich, St. Louise, MO, USA), radioimmunoprecipitation assay (RIPA) buffer (89900; Pierce), bicinchoninic acid assay (23225; ThermoFisher, Waltham, MA, USA), sample buffer (125 mM Tris pH 6.8, 4% sodium dodecyl sulfate (SDS), 10% glycerol, 0.006% bromophenol blue, and 1.8% mercaptoethanol), precast protein gel (4561094; Bio-Rad, Hercules, CA, USA), primary antibodies, anti-c-Met (ab51067; Abcam, Cambridge, UK; 4560; Cell Signaling, Massachusetts, USA), anti-nucleolin (ab22578/ ab13541; Abcam), anti-HER2 (SC-08; Santa Cruz, TX, USA), anti-GAPDH (SC-47724; Santa Cruz), MTS assay solution (G3585; Promega, WI, USA), μ-Slide 8-well (ibid; München, Germany), Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Labs), and Matrigel (BD Biosciences, NJ, USA) were used.

Protein extraction was performed using Ripa Buffer and Protease/Phosphatase Inhibitor Cocktail (Cell Signaling) and protein quantification using the Bradford method. Protein samples (30 μg) were mixed 1:1 with Laemmli buffer supplemented with 20% 1,4-Dithiothreitol (DTT) (Sigma) and loaded on 4–12% Tris-Glycine gels (Lonza). Running was performed at 125 V for 80′ and wet transfer at 25 V for 120′. Membranes were blocked with 5% milk in TBS-t for 1 h, at room temperature (RT). The primary Abs used were: anti-caspase-1 (2225, Cell Signaling), anti-ASC (AG-25B-0006, Adipogen) and anti-β-tubulin (2128, Cell Signaling). The secondary Abs were: peroxidase goat anti-rabbit IgG (H + L) (PI-1000) and peroxidase horse anti-mouse IgG (H + L) (PI-2000, Vector Laboratories). For detection we used ECL Select Reagent (Amersham, GE Healthcare), ChemiDoc™ MP system and Image Lab Software (Bio-Rad).

UltraPureTM Tris was purchased from Invitrogen (Auckland, New Zealand). Promega sequencing grade modified trypsin (PMV5111) was purchased from In Vitro Technologies (Auckland, New Zealand). Thiourea, hydrochloric acid, Tris(2-carboxyethyl)phosphine (TCEP), ammonium bicarbonate, hydrochloride solution trichloroacetic acid (TCA), urea, iodoacetamide, and 1,4-dithiothreitol (DTT) were purchased from Sigma Aldrich (St. Louis, MO, USA). Formic acid was from Scharlau (Barcelona, Spain). The EZQ^® Protein Quantitation Kit was from Invitrogen (Auckland, New Zealand). Acetonitrile, acetone and HPLC-grade methanol were from Merck (Darmstadt, Germany). Solid-phase extraction (SPE) cartridges (Strata-X, 33 μm Polymeric Reversed Phase, 10 mg/mL) were purchased from Phenomenex New Zealand (Auckland, New Zealand).

Formic acid (FA) for mass spectrometry (cat. #94318), sodium deoxycholate (SDC) BioXtra ≥ 98.0% (cat. #30970), ammonium bicarbonate (AmBic) BioUltra ≥ 99.5% (cat. #09830), and iodoacetamide (IAA) ≥ 99.0% (cat. #I6125) were all purchased from Sigma Aldrich (St. Louis, MO); 1,4-dithiothreitol (DTT) ≥ 99.0% (cat. #6908, Carl Roth, Karlsruhe, Germany); Trypsin Gold, Mass spectrometry grade (cat. #V5280, Promega, Madison, WI); LC–MS grade acetonitrile (ACN, cat. #1207802BS, Biosolve, Valkenswaard, The Netherlands). Water was produced using Millipore Simplicity 185 ultrapure water system (Merck Millipore corp. Billerica, MA).

Alcohol dehydrogenase (ADH), κ-casein, catalase and insulin were obtained from Sigma Chemical Co. (St. Louis, MO, USA). 1,4-dithiothreitol (DTT) and thioflavin T (ThT), were also purchased from Sigma. All other chemical reagents were purchased from AJAX Finechem (Cheltenham, VIC, Australia). Clear and μClear 96-well plates were obtained from Greiner Bio-One (Kremsmünster, Austria). ThinSeal was purchased from Excel Scientific Inc. (Victorville, CA, USA). Supor (0.2 μm) syringe filters and Macropep 10 kDa spin tubes were obtained from Pall Life Sciences (Port Washington, NY, USA). Formvar and carbon coated nickel electron microscopy grids were purchased from Pro Sci Tech (Kirwan, QLD, Australia).
Bovine eye lenses were supplied by local abattoirs (T. & R. Pty. Ltd., Murray Bridge, South Australia and CMP Canterbury Ltd., Ashburton, New Zealand). Escherichia coli containing the plasmid vector pET20b(+) with the human wild type αB-crystallin gene previously inserted was obtained from Prof. Wilbert Boelens, Radboud University, The Netherlands.