Dl dithiothreitol dtt

PCASMCs were cultured after isolation by enzymatic digestion from porcine small coronary arteries [41 (link)]. In brief, small coronary arteries were cut open longitudinally and the endothelium was gently scraped off with a microsurgical blade. The tissue strip was then washed with cold Krebs and dissected into 1×1 mm² strips, followed by 30-min digestion at 37°C in 2 ml of phosphate-buffered saline solution (PBS), containing 2 mg/ml of collagenase (type 2; Worthington Biochemicals, USA), 0.5 mg/ml of papain (Worthington Biochemicals, USA), 1.75 mg/ml of DL-Dithiothreitol (DTT, Sigma, USA), and 5 mg/ml albumin bovine (BSA, Amresco, USA). The enzymatic activity was stopped by Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific, USA) containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific, USA). The suspension was centrifuged at 1600 rpm for 5 min. Cells were then resuspended in 12 ml DMEM containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin (Thermo Fisher Scientific, USA). After 1-h incubation at 37°C, the medium was replaced once to remove unattached cells. Attached PCASMCs were cultured in a humidified incubator with 5%CO₂ at 37°C. For maintaining electrophysiological properties of isolated PCASMCs, only primary cultured cells were used for experiments.

The chemicals and reagents used in this work are the following: water (LC-MS grade-LiChrosolv^®) (Merck KGaA, Darmstadt, Germany), acetonitrile (LC-MS grade-LiChrosolv^®) (Merck KGaA, Darmstadt, Germany), 2-propanol (LC-MS grade-LiChrosolv^®) (Merck KGaA, Darmstadt, Germany), ammonium bicarbonate (NH₄HCO₃) (Sigma-Aldrich, Buchs, Switzerland), RapiGest^TM SF surfactant (Waters, Milford (MA), USA), DL-dithiothreitol (DTT) (Sigma-Aldrich, Buchs, Switzerland), iodoacetamide (IAA) (Sigma-Aldrich, Buchs, Switzerland), trypsin (porcine pancreas) (Sigma-Aldrich, Buchs, Switzerland), trifluoroacetic acid (TFA) (Honeywell SC, Seeelze, Germany), formic acid (FA) LiChropur^® (Merck KGaA, Darmstadt, Germany), Pierce^TM HeLa protein digest standard (Thermo Scientific^TM, Waltham, MA, USA) and MMI-L low concentration tuning mix (Agilent Technologies, Santa Clara, CA, USA).

Detection of plasma membrane LMTK2 was performed by domain selective plasma membrane biotinylation, as previously described (Swiatecka-Urban et al., 2002 (link)). CFBE41o- cells were treated with TGF-β1 or vehicle for 1 h. Apical or basolateral plasma membrane proteins of ALI cultures of HBE or CFBE41o- cells were selectively biotinylated using cell membrane impermeable EZ-Link^TM Sulfo-NHS-LC-Biotin (Pierce Chemical Co., Dallas, TX, United States), followed by cell lysis in buffer containing 25 mM HEPES, 10% v/v glycerol, 1% v/v Triton X-100, and Complete Protease Inhibitor Mixture (Roche Applied Sciences, Indianapolis, IN, United States). Lysates were centrifuged at 14,000 x g and biotinylated proteins were isolated from the supernatants, considered as whole cell lysates (WCL), by incubation with streptavidin-agarose beads, eluted into 2x Laemmli sample buffer (Bio-Rad Laboratories) containing DL-dithiothreitol (DTT; Sigma-Aldrich), and separated by 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad Laboratories). The immunoreactive bands were visualized by western blotting (WB) with Western Lightning Chemiluminescence Reagent Plus (PerkinElmer LAS Inc., Boston, MA, United States). Protein abundance was quantified by densitometry using exposures within the linear dynamic range of the film.

Colonic lamina propria cells were isolated as detailed.³⁵ Briefly, animals were euthanised as previously described, and the large intestine was dissected, longitudinally opened, washed with PBS and cut into small pieces. Tissue fragments were placed in Petri dishes and washed three times in calcium- and magnesium-free HBSS containing 1 mM dl-dithiothreitol (DTT, Sigma-Aldrich, Irvine, UK) for 30 min. Supernatants were discarded. After that, tissue fragments were incubated with 100 µl/mL of collagenase II (Sigma-Aldrich) for 60 min at 37 °C on a shaker. Supernatants were passed through a 70-µm cell strainer (Falcon, Corning, NY, USA), and then resuspended in R-10 medium (RPMI with 10% FBS, 2 mM L-glutamine (Thermo Fisher), 1 mM sodium pyruvate (Thermo Fisher), 1% non-essential amino acids (Thermo Fisher), 1% Pen/Strep, 1% vitamin solution (Thermo Fisher) and 5 × 10⁻⁵ M 2β-mercaptoetanol) for further analysis.

The P. lividus were collected from October to May in the Gulf of Naples and maintained at 16 °C in circulating seawater. The eggs were spawned by injecting 0.5 M of KCl into the body cavity. The released eggs were collected in natural seawater (NSW, pH 8.1) filtered with a Millipore membrane of 0.2 µm pore size (Nalgene vacuum filtration system, Rochester, NY, USA) and used for the experiments in the next 3 hr. The dry sperm were collected by pipetting from the male animal’s body and kept at 4 °C. A few minutes before insemination, the sperm were diluted in NSW at a final concentration of 1.84 × 10⁶ units/mL. In the experiments where dejellied eggs were used, the egg jelly was removed by placing the eggs in seawater titrated with HCl to pH 5.5 for 5 min. The eggs incubated in 1 mL of seawater at pH 5.5 or after washing with NSW were fertilized with 10 µL of sperm diluted in NSW. The removal of the VL was performed by incubating the eggs for 20 min in NSW containing a final concentration of 10 mM of DL-Dithiothreitol (DTT) (Sigma-Aldrich, St. Louis, MO, USA) at pH 9 adjusted with 1 mM of NaOH [26 (link)]. When needed, the eggs suspended in acidic seawater or after the removal of the VL were transferred to NSW for the fertilization experiments.