Nonidet p 40 substitute np 40

Dulbecco’s Modified Eagle Medium (DMEM) powder was purchased from Gibco by Life Technology (Thermo Fisher Scientific, Waltham, MA, USA). Calcium chloride dihydrate (CaCl₂·2H₂O), sodium bicarbonate (NaHCO₃), sodium hydrogen phosphate (Na₂HPO₄), potassium dihydrogen phosphate (KH₂PO₄), sodium citrate tribasic dihydrate, sodium succinatedibasic hexahydrate and trypsin-ethylene diaminetetraacetate (EDTA) salts were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dimethyl sulphoxide (DMSO), thiazolyl blue tetrazolium bromide (MTT), nonidet P40 substitute (NP40) and anti-cancer drug DOX were acquired from Sigma-Aldrich (St. Louis, MO, USA). DMEM liquid media, foetal bovine serum (FBS), TrypLE Express, and penicillin-streptomycin were procured from Sigma-Aldrich (St. Louis, MO, USA). Sodium chloride and potassium chloride salts were bought from Fischer Scientific (Loughborough, UK). Acetonitrile (ACN), hydrochloric acid (HCl), methanol and glycerol were from Fischer Scientific (Loughborough, UK). DOX was dissolved in distilled water and 8.62 mM stock solution was prepared.

N-Glycans were released from the protein fraction as previously described [30 (link)]. To this end, 5 μL of plasma were denatured with 10 μL 2 % sodium dodecyl sulfate (SDS; Merck, Darmstadt, Germany) and incubated for 10 min at 60 °C. The subsequent release step was performed by adding 10 μL of a mixture containing 2 % Nonidet P-40 substitute (NP-40; Sigma-Aldrich) and 0.5 mU recombinant peptide-N-glycosidase F (PNGase F; Roche Diagnostics, Mannheim, Germany) in 2.5× PBS, followed by overnight incubation at 37 °C. In case of peritoneal fluid, 100 μL of sample were dried by vacuum centrifugation and solubilized in 10 μL Milli-Q (MQ) water followed by 10 min sonication. The denaturing step was performed by adding 20 μL 2 % SDS for 10 min at 60 °C. The N-glycans were released by adding 20 μL of a mixture containing 2 % NP-40 and 1 mU PNGase F in 2.5× PBS, followed by overnight incubation at 37 °C.

One 10 cm petri dish of cells transfected as described above was used for each sequential extraction of proteins with buffers of increasing stringency. Cells were harvested by scraping in solubilization buffer (SB: 10 mM Tris pH 7.4, 150 mM NaCl, 0.5 mM EDTA, 1 mM DTT, complete EDTA-free protease inhibitors (Roche). Total protein concentrations were measured and 1 mg of total protein was used for the SIA. Samples were treated with Benzonase (250 units per sample in 2 mM MgCl₂, 30 min at 4 °C (Roche)) before, and at each solubilization phase of SIA. For each step, samples were centrifuged for 30 min at 16,000×g at 4 °C. After removing the supernatant (soluble material), pellets (insoluble material) were suspended in SB 1% v/v Nonidet P40 Substitute (NP-40, Sigma), SB 2% w/v dodecyl-ß-d-maltoside (Sigma), SB 2% w/v N-Lauroylsarcosine (Sarkosyl, Sigma) and SB 1% w/v sodium dodecyl sulfate (SDS, Sigma) sequentially. SDS insoluble material was suspended in SB 1x SDS blue loading buffer (Life Technologies).