Ecl plus

Tissue and cell samples were prepared in lysis buffer containing 1 mM PMSF (Millipore Sigma, MA, USA). Pierce BCA protein assay kit (Thermo Fisher Scientific, MA, USA) was used to calculate the protein concentration. Equal amounts of proteins or plasma (2.0 μl) were separated by denaturing SDS-PAGE. Proteins were transferred onto PVDF membranes (Bio-Rad, CA, USA) and probed with the primary antibody followed by incubation with the HRP-conjugated secondary antibody. ECL™ Plus or ECL™ Prime system (GE Healthcare, IL, USA) was used to detect protein signal. The expression level was determined by measurement of the corresponding band intensities by using ImageJ software, and the relative values of the phosphorylated protein were expressed relative to total protein signal. Blood sample was collected by heart puncture from mice that were fasted for 12 h (Maruyama et al., 2012 (link)).

For immunological detection, 20 μg of cell lysate was separated using SDS-PAGE (7.5–10% Tris gel). Then, the proteins were blotted onto a PVDF membrane, which was incubated with the PVDF blocking reagent Can Get Signal (TOYOBO, Osaka, Japan). Proteins were probed with primary antibodies against β-catenin (1/1000, Abcam, Cambridge, MA, USA), FOXF2 (1/1000, Abnova, Walnut, CA, USA), WNT2B (1/2000, Abcam), LMNB1 (1/2000, Proteintech, Rosemont, IL, USA), and GAPDH (1/2000, MBL, Aichi, Japan). A horseradish peroxidase-conjugated goat anti-rabbit antibody was then added, and secondary antibodies were detected through autoradiography using enhanced chemiluminescence (ECL Plus, General Electric Healthcare, Chicago, IL, USA).

Pulmonary arteries collected from 22 rats were used in Western blot analysis, one sample was missing due to technical issues. Total protein content in pulmonary arteries was quantified using the bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Massachusetts, USA). Samples were measured at 562 nm (PHERAstar, BMG Labtech, Ortenberg, Germany) after 30 min incubation at 37 °C. The supernatant was used to prepare gels while in Laemmli sample buffer containing 2% SDS. 12% Criterion Precast Gel (Bio-Rad laboratories, Copenhagen, Denmark) was used to separate proteins, which were then electrotransferred to a nitrocellulose membrane. Blots were blocked with 5% nonfat dry milk in PBS-T as described previously^{49 (link)}. After washing with PBS-T, blots were incubated with primary antibodies (COX-1, COX-2 and beta-actin) overnight at 4 °C, and visualized with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature using the enhanced chemiluminescence system (ECL Plus, GE Healthcare, Amersham, United Kingdom). Values were normalized to total protein using Stain-Free technology.

Western blot analyses were performed with iHEK cell lysates. Approximately 10 µg of proteins preparations were loaded on precast NuPAGE 4–12% Bis–Tris gels (Invitrogen) for sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were subsequently transferred onto nitrocellulose membranes and blocked overnight in 10% nonfat dry milk at 4 °C. Membranes were then probed for 1 h at RT using primary antibodies against α-MSI1 (1:1000, Abcam), α-MSI2 (1:1000, Abcam) and Pan-Tau (1:10,000, Tau13), GAPDH (1:1000), LaminB1 (1:1000) and Histone3 (1:1000) diluted in 5% nonfat dry milk. α- MSI1 and α-MSI2 immunoreactivities were detected with a horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:6000, GE Healthcare). Immunoreactivity for other antibodies was detected using an anti-mouse IgG (1:6000, GE Healthcare) diluted in 5% nonfat milk. Enhanced chemiluminescence (ECL) plus (GE Healthcare) was used for band visualization. LaminB1 and GAPDH were probed to normalize and quantify nuclear and cytoplasmic proteins, respectively. Compartment extraction was conducted with Qproteome Cell Compartment Kits (Qiagen, #37502), and nuclear, cell membrane, and cytoplasmic proteins were isolated and preserved for western blot analysis.

As described (9 (link), 10 (link), 16 (link)), breast cancer cell lines were grown to log-phase, washed twice with ice-cold PBS, and lysed in SDS lysis buffer according to the manufacturer's protocol (Cell Signaling Technology, Danvers, MA). Protein concentration was quantified using the bicinchoninic acid reagent (Sigma). Cell lysates containing 25 μg of protein were separated by SDS-polyacrylamide gel electrophoresis (PAGE), and transferred to PVDF membranes (Millipore). Membranes were immunoblotted with antibodies against phospho-AKT (S473), total AKT, phospho-p70S6K (T389), total p70S6K, phospho-rpS6 (S235/ 236), total rpS6, phospho-ERK (T202/Y204), total ERK (all from Cell Signaling Technology), tubulin (Sigma), and actin (Millipore). Bound antibody was visualized using SuperSignal West Pico (Thermo Scientific, Waltham, MA) or ECL plus (GE Healthcare, Auckland, NZ) and the chemiluminescence detection system by Fujifilm Las-3000.

Dot blot analysis was performed as previously described [7 (link)]. Briefly, isolated DNA was denatured and then serially diluted twofold. DNA samples were spotted on a nitrocellulose membrane using a Bio-Dot apparatus (Bio-Rad) and immunoblotted with rabbit anti-5-hydroxymethylcytosine polyclonal antibody (Active Motif, 1:5000) in Odyssey:PBS (Li-Cor). The membrane was washed and then incubated with HRP-conjugated goat anti-rabbit IgG (Abcam, 1:10,000) secondary antibody in Odyssey:PBS. The membrane was visualized by chemiluminescence with GE ECL Plus.

For immunological detection, 20 μg of cell lysate was separated via SDS-PAGE (7.5–10% Tris gel). After the proteins were blotted onto a PVDF membrane, the membrane was incubated with the PVDF blocking reagent Can Get Signal (TOYOBO). Proteins were probed with primary antibodies against CDK1 (MBL) and GAPDH (MBL) and secondary antibodies were detected through autoradiography using enhanced chemiluminescence (ECL Plus, General Electric Healthcare).