Phosstop solution

Frozen tissues were homogenized with an ice‐cold RIPA buffer. RIPA buffer contained 50 mmol/L Tris‐HCl pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA, 0.1% SDS, 1% NP40, 0.25% sodium azide, with 10 μL protease inhibitor cocktail (Sigma, P8340), 1 mmol/L phenylmethylsulfonyl fluoride (PMSF–Sigma, P7626), and 1 mL PhosStop solution/10 mL RIPA (PhosStop – Roche, 4906837001). Protein concentration was measured with the Bradford reagent (Sigma, B6916). Western blot analysis was performed as described (Rim et al. 2005; Anunciado‐Koza et al. 2008; Chu et al. 2014). The blots were incubated with antibodies against SCL25A1 (rabbit anti‐Scl25a1 – Santa Cruz Biotech sc‐86392) (1:200), ACLY (Rabbit anti‐Acly, Abcam, ab40793) (1/2000), MEST (Rabbit anti‐MEST (Nikonova et al. 2008)) (1:700), and β‐actin (mouse anti‐β‐actin – Abcam, Ab6276) (1:10000). Specific antibody–antigen complexes were detected using fluorescent‐labeled secondary antibodies (goat anti‐rabbit IRDye 800, Rockland, 611‐132‐122; goat anti‐mouse IRDye 800, Rockland, 610‐730‐124; Donkey anti‐goat IRDye 700, Licor, 926‐32214 and Donkey anti‐mouse IRDye 700, Rockland, 610‐730‐124). Bands were visualized and quantified using the Odyssey imaging system (Licor Bioscience). β‐actin was used as an internal control to adjust for variability in protein loading and transfer.

Cells were washed twice with PBS, harvested, and then lysed in RIPA buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS)) containing complete mini and phosSTOP solution (Roche). After sonication and removal of debris by centrifugation, 20 μg protein from each sample was resolved by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were immunoblotted with the indicated antibodies: cleaved TRPV2 (1:250) and β-actin (1:500). Antigens were revealed by Immobilon Western HRP Substrate (Millipore, Billerica, MA, USA) after incubation with horseradish peroxidase-conjugated anti-rabbit IgG and visualized by enhanced chemiluminescence (Amersham Biosciences, Buckinghamshire, UK). Band densities were quantified by using Image J software. For in vivo analysis, alveolar macrophages collected by BAL from five mice were pooled because the yield of alveolar macrophages by BAL is very few.

Embryos were fixed in 4% PFA (30 min, RT), permeabilised with 0.5% Triton-X100 (30 min, RT) and blocked with 3% BSA. Plk1 phosphorylated at Thr210 was labeled with a polyclonal rabbit antibody (ThermoFisher Scientific; dilution 1:100 in 3% BSA, overnight, 4 °C) and cyclinA2 - with a polyclonal rabbit antibody (Santa Cruz; dilution 1:50 in 3% BSA, overnight, 4 °C), both followed by a secondary anti-rabbit antibody conjugated with Alexa 633 (Molecular Probes, ThermoFisher Scientific; dilution 1:200 in 3% BSA, 1.5 h, RT). Microtubules were stained with mouse anti-tubulin β antibody labeled with FITC (Sigma-Aldrich; dilution 1:50 in 3% BSA, 1.5 h, RT or overnight, 4 °C). DNA was stained with Hoechst 33342 dye (Molecular Probes, ThermoFisher Scientific; 100ng/µl in PBS, 30 min, RT or overnight, 4°C), propidium iodide (Sigma-Aldrich; 3 μg/ml in PBS, 30 min, RT) or chromomycin A3 (Sigma-Aldrich; 10 μg/ml, 30 min, RT or overnight, 4°C). In case of phospho-Plk1 detection, a PhosStop solution (Roche) was added to all stages of immunostaining to inhibit phosphatases.