1 4 dithioerythritol

Third‐order mesenteric arteries (n = 3 animals as separate experiments) were cleared of attached fat and incubated in HEPES‐buffered physiological solution (NaCl 154 mM, KCl 5.4 mM, MgSO₄ 1.2 mM, glucose 10 mM, CaCl₂ 10 mM and HEPES 10 mM; pH 7.4) supplemented with 1,4‐dithioerythritol (Sigma‐Aldrich, Gillingham, UK) at 2 mg/ml and papain (Worthington Biochemical, Lakewood, New Jersey, USA) at 1 mg/ml in a water bath at 37°C for 22 min. The solution was then replaced with HEPES buffer supplemented with type F collagenase from Clostridium histolyticum (Sigma‐Aldrich, Gillingham, UK) at 1 mg/ml for 7 min. The cell suspension was briefly stood on ice before being gently titrated and small drops of cell suspension pipetted onto poly‐l‐lysine‐coated slides (VWR International, Radnor, Pennsylvania, USA). Slides were left at room temperature for 50 min. before ice‐cold methanol was added to the cell suspension for 10 min. Slides were carefully washed in HEPES buffer before being stored under saline solution at 4°C before being probed for PMCA4 as described below.

Deionized water was obtained by Milli-Q Element water-purification system (Millipore, Bedford, MA, USA). Urea was purchased from VWR International (Milan, Italy). Hexane (≥ 99%), trypsin sequence-grade, 1,4-dithioerythritol (DTE), iodoacetamide (IAA), acetonitrile and formic acid were purchased from Sigma Aldrich (Milan, Italy).