0.2 μm pvdf membrane

As described previously [23 (link)], cells were lysed in Glo Lysis Buffer (Promega) supplemented with protease inhibitor and phosphatase inhibitor cocktails (Roche). Next, 20–60 μg of total proteins (as determined by BCA assay kit) was resolved by 10% or 12% SDS-PAGE gel, transferred onto a 0.2-μm PVDF membrane (Millipore, CA, USA), and probed with antibodies as indicated in the figures.

Proteins were extracted from ground lung tissue or cultured cell samples by adding RIPA lysate (Solarbio Life Science, China). The protein concentration was determined with a PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) after an addition of a phosphatase inhibitor and a protease inhibitor (APExBIO Technology LLC, Houston, TX, USA) according to the volume of the sample. Proteins were heated to 100 °C for denaturation and then electrophoresed in 10% SDS-PAGE gels. The proteins were transferred with a 0.2 μm PVDF membrane (Millipore, Burlington, MA, USA) and blocked with 5% skimmed milk (Sigma-Aldrich, USA) for 2 h. After elution, the samples were incubated with the corresponding primary antibody at 4 °C overnight. After 1 h of secondary antibody (Proteintech, Shanghai, China) incubation at room temperature, the proteins were visualized with ECL luminescent solution (Cwbio, Taizhou, China) in a GeneGnome XRQ imager (Syngene, Cambridge, UK). The gray values of the bands were calculated and analyzed using ImageJ 1.5.2a software. The primary antibodies used in this experiment are shown in Table 1.

GC-1 cells stably expressing either ANKRD49 or ANKRD49 shRNA at 80% confluence were treated with serum-free media for 24 h as starvation treatment; the NF-κB pathway inhibitors, pyrrolidine dithiocarbamate (PDTC) (50 μM) (Sigma, USA) or BAY 11–7082 (10 μM) (Santa Cruz, USA) was added to complete media for 2 h prior to the serum-free media treatment. Nuclear proteins were obtained using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce, USA), total proteins were extracted with RIPA buffer and quantified using the BCA protein assay reagent (Thermo Scientific, USA). Protein samples were separated by 12% SDS-PAGE and transferred to a 0.2 μm PVDF membrane (Millipore, USA). Membranes were blocked in 5% skim milk for 1 h at room temperature, followed by an overnight incubation at 4°C with primary antibody followed by incubation with their corresponding HRP-IgGs, then visualized using an ECL blot detection system (Transgene, Beijing, China). Band intensities were quantified using a Tanon 1600 Gel Image Analysis System.

Total protein samples were extracted from snap-frozen liver tissues (80–100 mg) of different groups using 1 ml RIPA lysis buffer (Pierce, Rockford, Illinois, USA) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, St. Louis, Missouri, USA), a protease inhibitor cocktail (Amresco, Solon, Ohio, USA) and phosphostop (Roche Diagnostics, Indianapolis, Indiana, USA). Total extracts were collected by centrifugation at 14000× g for 10 min at 4 °C. The concentration was determined using a BCA protein assay kit (Pierce, Rockford, Illinois, USA). Then protein samples were subjected to SDS-PAGE (15%) and transferred onto a 0.2-μm PVDF membrane (Millipore, Darmstadt, Germany). Membranes were blocked with 5% skimmed milk and incubated with specific primary antibodies against p65 (1:1000), α-SMA (1:1000), TGF-β1 (1:1000) and β-actin (1:10000) overnight at 4 °C. Membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit or goat anti-mouse secondary antibody (1:20000) at room temperature for 1 hour. At last, protein bands were visualized using Immobilon enhanced chemiluminescence (ECL) reagents and imaged using a GelDoc XR System (Bio-Rad, Shanghai, China). Bands densities were analyzed using Image J free software (http://rsb.info.nih.gov/ij/).

To obtain cell suspensions, embryogenic cultures were multiplied and maintained in the basic liquid culture medium MSG [38 ] supplemented with 30 g l^-1 sucrose, 1.4 g l^-1 L-glutamine (Sigma-Aldrich, St. Louis, USA), and 0.1 g l^-1 myo-inositol (Merck KGaA, Darmstadt, Germany), and the pH of the culture medium was adjusted to 5.7 before autoclaving at 121°C for 20 min, 1.5 atm. The embryogenic cell suspension cultures were subcultured every 15 days by adding 10 ml of the old suspension culture to 60 ml of fresh liquid medium. Embryogenic cell suspension cultures were kept on an orbital shaker (Cientec, Minas Gerais, Brazil) at 100 rpm in the dark, at 25 ± 2°C.
To analyze the effect of Mps1 inhibition on cellular growth and the PEM morphology, embryogenic cell suspension cultures were grown in basic MSG culture medium supplemented with 30 g l^-1 sucrose, 1.4 g l^-1 L-glutamine, and 0.1 g l^-1 myo-inositol, and with or without Mps1 inhibitor SP600125 (Sigma-Aldrich). The Mps1 inhibitor was filter-sterilized through a 0.2-μm PVDF membrane (Millipore, São Paulo, Brazil) before being added to the culture medium. After the inoculation of embryogenic cell culture with 15-day-old cell suspensions, the flasks were maintained on an orbital shaker at 100 rpm in the dark, at 25 ± 2°C.

For total protein extraction, cells were cultured in 12-well plates and lysed in cell lysis buffer (150 mM NaCl, 1% NP-40, 50 mM Tris–HCl pH 8.0, cOmplete EDTA-free Protease Inhibitor Cocktail (#11,836,170,001) and PhosSTOP phosphatase inhibitor tablets (#PHOSS-RO) purchased from Roche), sonicated (15 cycles: 15 s on, 30 s off) with a Diagenode Bioruptor sonication system at 4 °C, and then centrifuged 20 min at 15,000 g. The supernatants were collected and boiled at 95 °C for 5 min with sample buffer (0.25 M Tris–HCl pH 6.8, 50% glycerol, 5% SDS, 0.05% bromophenol blue, and 10% 2-mercaptoethanol). The samples were then run on 4–12% gradient glycine gels (#NP0321BOX) or 16% tricine gels (#EC6695BOX) purchased from Thermo Fisher Scientific and blotted onto a 0.2 μm PVDF membrane purchased from Millipore (#ISEQ00010). The membranes were blocked with 3% BSA in PBS-T (0.1% Tween 20 in PBS) for 1 h at room temperature, and then stained overnight at 4 °C with 3% BSA in PBS-T and the primary antibodies referenced above. Membranes were washed with PBS-T and stained for 1 h at room temperature with fluorescent secondary antibodies: anti-mouse IgG(H + L) DyLight 800 conjugate (#5257, CST) and anti-rabbit IgG(H + L) DyLight 680 conjugate (#5366, CST). The images were captured using a LiCor Odyssey Infrared Imaging System 9120 (LI-COR Biosciences).