Mini edta free protease inhibitor cocktail tablet

Pichia pastoris wild type strain X-33 (Invitrogen) was electroporated to integrate the hCD81 expression plasmid comprising pPICZB encoding C-terminus His₆ tagged CD81 [20 (link)]. SMA 2000 co-polymer (2:1 Cray Valley) and SZ25010 co-polymer (3:1 Polyscope) were obtained as styrene maleic anhydride and hydrolysed to styrene maleic acid forms as described previously [21 (link)]. Detergent n-dodecyl-B-D-maltoside (DDM), biotin, zeocin, yeast extract, peptone, yeast nitrogen base and agar antibiotic were purchased from ThermoFisher Scientific. Ni²⁺-NTA resin for IMAC purification was purchased from Qiagen and Bio-Rad Econo-column empty chromatography column was used. Mini EDTA-free Protease Inhibitor Cocktail Tablets from Roche Applied Science. Anti-mouse HRP-conjugated secondary antibody was purchased from Cell Signalling Technology. Ammonium formate was purchased from Sigma Aldrich. Methanol, chloroform and ultra pure water, all LC-MS grade, were purchased from Fisher, UK. Anti-CD81 antibodies 2s131, 1s337 and 1s135 were generated as described previously [22 (link)] and hybridoma supernatants were used for all ELISA experiments.

Total protein was isolated from cultured cells using cOmplete Lysis-M EDTA-free and cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail Tablets (Roche, Basel, Switzerland). Protein concentrations were assessed using a BCA protein assay kit (Thermo Fisher Scientific). Equivalent amounts of protein samples were mixed with NuPAGE LDS Sample Buffer (4×), separated in NuPAGE Novex 4–12% Bis-Tris Gels (Life Technologies), and transferred onto nitrocellulose membranes. The membranes were blocked with blocking buffer (TBS-T [25 mM Tris-HCl pH 7.4, 125 mM NaCl, 0.05% Tween-20] with 5% bovine serum albumin [Sigma-Aldrich, St. Louis, MO, USA]) for 1 h, and incubated overnight at 4°C with the following primary antibodies and subsequently with corresponding secondary antibodies for 1 h at room temperature. Antibodies and dilutions used were rabbit monoclonal anti-E-cadherin (1:1000, Cell Signaling Technology, Danvers, MA, USA), goat polyclonal anti-Snail (1:1,000; Abcam, Cambridge, UK), rabbit monoclonal anti-N-cadherin (1:1,000; Cell Signaling Technology), rabbit monoclonal anti-vimentin (1:1,000; Cell Signaling Technology), and mouse monoclonal anti-β-actin (1:5,000; Santa Cruz Biotechnology, Dallas, TX, USA). Immunoreactive bands were detected using the ChemiDoc XRS + Imaging System (Bio-Rad Laboratories), and protein levels are presented relative to that of β-actin.

Proteins extracts enriched in GPI-anchored proteins were resuspended in 10 µL of a solution containing 0.5% SDS and 40 mM DTT and denatured for 10 min at 100 °C. Samples were allowed to cool and the reaction volume completed to 20 µL in 50 mM of sodium citrate pH 5.5 containing 500 U of endoglycosidase H (Endo H, New England Biolabs). The reactions were carried out at 37 °C for 3 h followed by inactivation at 75 °C for 10 min. Samples were resolved on 10% SDS-PAGE and the extent of deglycosylation analysed by immunoblotting using 3F6 and 9E10 monoclonal antibodies. For deglycosylation of total parasite extracts, epimastigotes were washed thrice with PBS and adjusted to a concentration of 1 × 10⁸ parasites/mL in PBS supplemented with complete Mini EDTA-free Protease Inhibitor Cocktail Tablets (Roche) and 1 mM PMSF. Aliquots containing 5 × 10⁶ cells were frozen and concentrated to dryness in SpeedVac (Heto). Samples were resuspended and processed as aforementioned.

Kidney samples were homogenized in RIPA buffer with a protease inhibitor mix (complete™, Mini, EDTA-free Protease Inhibitor Cocktail Tablets, Roche, Basel, Switzerland), employing the TissueLyser LT to promote lysis. Total protein content of each sample was measured by the BCA Protein Assay Kit. Mini-PROTEAN® precast gel 4–12% (Bio-Rad, Milan, Italy) was operated for loading the samples and Precision Plus Dual Color Standards (Bio-Rad, Milan, Italy) was used as molecular weight marker. Trans-Blot® Turbo Nitrocellulose membrane (Bio-Rad, Milan, Italy) was employed to transfer proteins. In order to detect the oxidative damaged proteins in the samples, the primary antibody anti-NOX4 (1:1,000, Rabbit polyclonal antibody, Abcam, Milan, Italy) was used. Anti-GAPDH (1:20,000, Rabbit monoclonal antibody, OriGene, Herford, Germany) antibody was used as housekeeping expression protein. Nitrocellulose membranes were incubated with HRP-labeled secondary antibodies (1:2,000, Santa Cruz Biotechnology, Heidelberg, Germany) according to the species of primary anti-bodies and bands were detected using Clarity Western ECL substrate (Bio-Rad, Milan, Italy). Relative signal intensities were quantified by ChemiDoc™ Imaging System (Bio-Rad, Milan, Italy) with the Bio-Rad Quantity One® software version 4.6.3. and normalized in relation to GAPDH bands.

Enrichment of GPI-anchored proteins was performed as described by Cordero et al.^{45 (link)}. Briefly, parasites were washed thrice with PBS and lysed on ice in a solution containing 10 mM Tris-HCl, 150 mM NaCl containing 2% of TX-114 (TBS-2% TX-114) supplemented with complete Mini EDTA-free Protease Inhibitor Cocktail Tablets (Roche) and 1 mM PMSF. Samples were clarified at 8,800 × g and the supernatant incubated at −20 °C for 24 h. Samples were thawed and submitted to 4 consecutive temperature-induced phase separations at 37 °C. Samples were clarified at 18,000 × g for 10 min at 0 °C and submitted to a final phase separation at 37 °C. The final detergent-rich phase was precipitated with 3 volumes of cold acetone and the proteins recovered by centrifugation at 18,000 × g. Aliquots were analysed by SDS-PAGE stained with colloidal Coomassie or silver nitrate and by immunoblotting after transfer onto nitrocellulose membranes.