Ab116028

Cells were lysed in RIPA buffer (supplemented with a protease and phosphatase inhibitor cocktail) and homogenates were solubilized in Laemmli buffer [61 (link)]. 15–30 μg solubilized proteins were subjected to 12.5% SDS-polyacrilamide gel electrophoresis and transferred to the Hybond-P PVDF Membrane (GE Healthcare, Chicago, IL, USA) by electroelution. The membranes were incubated overnight with anti-AQP3 antibody produced in rabbit (SAB5200111; Sigma, Italy) diluted 1:1000 in TBS and 0.1% Tween-20. The membranes were washed and incubated for 1 h with goat anti-rabbit IgG antibody, peroxidase conjugated (AP132P; Millipore, Burlington, MA, USA) diluted 1:100,000 in blocking solution. The bands were detected with ECL™ Select western blotting detection system (GE Healthcare). Pre-stained molecular weight markers (ab116028, Abcam) were used to estimate the molecular weight of the bands. Blots were stripped and re-probed with RabMAb anti β-2-microglobulin antibody ([EP2978Y] ab75853; Abcam, 1:10000) or with anti β-actin polyclonal antibody (Cat.n. AB-81599; Immunological sciences, USA, 1:2000) as housekeeping.

Yeast pellets were lysed and processed as described for Phos-tag Western blotting. The samples were loaded on home-made 6–14% acrylamide gradient Bis–Tris gels (0.33 M Bis–Tris pH 7.5) alongside broad molecular weight (10–245 kD) prestained protein ladder (ab116028; Abcam) and run at 130 V for 110 min using MES-SDS running buffer (Formedium MES-SDS5000). Gels were incubated in transfer buffer (NuPAGE Transfer Buffer with 20% ethanol final, NP0006; Thermo Fisher Scientific) and transferred onto 0.2-µm nitrocellulose membrane (1620112; Bio-Rad) at 25 V constant for 30 min using semidry transfer apparatus (1704150; Bio-Rad). Membranes were stained with Ponceau S solution (sc-301558; Santa Cruz) before imaging on a Bio-Rad ChemiDoc MP System to confirm equal protein loading. Membranes were then washed in Tris-buffered saline (TBS: 50 mM Tris–Cl, pH 7.5, 150 mM NaCl) and blocked in 5% nonfat dry milk in TBS. After further washes in TBS, membranes were incubated overnight at 4°C in primary antibody (diluted in 4% BSA, 0.02% sodium azide in TBS). After washing in TBS-T (TBS with 0.1% Tween-20), membranes were incubated in secondary antibody (diluted in TBS-T) for 1 h at room temperature, before washing again in TBS-T and developing using Clarity (1705061; Bio-Rad) or Clarity Max (1705062; Bio-Rad) ECL reagent on a Bio-Rad ChemiDoc MP System.

Cells were lysed in passive lysis buffer (Promega, E194A) and lysates were boiled at 98 °C for 5 min. The samples were cooled to room temperature and the insoluble debris was collected by centrifugation at (17,000 × g) for 1 min. Samples were then analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) alongside protein molecular mass markers (Abcam, ab116028). Proteins separated by SDS/PAGE were transferred onto nitrocellulose membranes (GE Healthcare) by using Trans-Blot Turbo transfer system (Bio-Rad) in 25 mM Tris⋅HCl, 250 mM glycine, and 20% methanol. Membranes were blocked with 5% skimmed milk in PBST or 5% BSA in TBST at 4 °C overnight. Blocked membranes were then incubated with primary antibodies overnight at 4 °C with consistent agitation, washed three times in PBST or TBST, and then incubated with secondary antibodies for 2 h at room temperature. The membranes were then washed three times with PBST or TBST, dried at room temperature, and imaged using the LI-COR Odyssey imaging system.