Supersep ace

To certify the primary structure of circulating SA in the dolphin, amino acid sequencing of the N-terminus of DSA was performed using sera from the two dolphins in Shinagawa Aquarium. After performing the albumin extraction using the method described in subsection above, the extract was analyzed using SDS-PAGE on a 10% polyacrylamide gel (SuperSepTM Ace, FUJIFILM-Wako), followed by transfer to a PVDF membrane using a Trans-blot TurboTM Transfer System (Bio-Rad Laboratories, Hercules, CA, United States). The membrane was washed with 50% methanol and Milli-Q water, and the section at around 66 kDa was excised. Sequencing of the initial five amino acids at the N-terminus was outsourced to the Institute for Protein Research in Osaka University (Suita, Osaka, Japan).

Proteins were extracted from the treated cells using cell lysis buffer (Cell Signaling Technology) containing a protease-inhibitor mixture (Thermo Scientific, Rockford, IL, USA) and phosphatase-inhibitor mixture (Nacalai Tesque, Kyoto, Japan). In some experiments, the nuclear and cytoplasmic fractions were extracted using NE-PER (Thermo Fisher Scientific) according to the manufacturer’s instructions. Protein concentrations were measured using a DC protein assay kit (Bio-Rad, Hercules, CA, USA). Equal volumes of protein sample were loaded on precast 12.5% polyacrylamide gel (SuperSepTM Ace; Fujifilm Wako Pure Chemical Co) and electrophoresed in Tris–glycine sodium dodecyl sulfate (SDS) running buffer. The subsequent analyses (blotting, antibody reaction and band detection) were conducted following previously reported protocols [47 (link)] with primary antibodies against Iκ-Bα, p65, phospho-JNK, JNK, phospho-p38 MAPK, p38 MAPK, phospho-ERK, ERK, phopho c-Jun, c-Jun and β-actin. The band intensity of each blot was quantified by densitometric analysis using Image LabTM^® 2.0 software (Bio-Rad).

HCT116 and U2OS cells were resuspended in benzonase buffer (20 mM Tris–HCl (pH 7.5), 40 mM NaCl, 2 mM MgCl₂, 0.5% NP-40, 50 U/ml benzonase, protease inhibitor cocktail, PhosSTOP (Roche, 4906845001), and incubated on ice for 10 min. NaCl (5 M) was then added to the samples at a final concentration of 450 mM. The samples were rotated at 4 °C for 30 min and then centrifuged at 13,000 rpm for 10 min. The supernatants were separated using 15% SuperSep Ace (Wako, 193-14991) and transferred onto PVDF membranes. The membranes were blocked with 5% skim milk and subsequently incubated with primary antibodies at 4 °C overnight. After overnight incubation, the membranes were incubated with AP-conjugated secondary antibodies and treated with a BCIP-NBT alkaline phosphatase solution (Nacalai, 03937-60) for detection. Images were cropped and processed using Photoshop 2020 (Adobe, USA).

Cultured cells were homogenized in RIPA buffer (Cell Signaling) containing protease inhibitors (Complete; Roche, IN, USA). Protein concentrations were measured using the Bradford protein assay (Bio-Rad), according to the manufacturer’s instructions. Protein samples (5 µg) was boiling for 5 min, then subjected to 13- or 17-wells of 5%–20% SDS-polyacrylamide Supersep Ace (Wako) gel electrophoresis. Gels that contains separated products were transferred to nitrocellulose membranes, followed by blocking with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20. Nitrocellulose membranes then incubate overnight with primary antibodies for p-FAK (Y397) (1:1000, Cell Signaling), FAK (1:1000, Cell Signaling), p-ERK1/2 (T202/Y204) (1:1000, Cell Signaling), ERK1/2 (1:1000, Cell Signaling), p-Akt (S473) (1:1000, Cell Signaling), Akt (1:1000, Cell Signaling), and GAPDH (1:1000, Proteintech, IL, USA). After washing with Tris-buffered saline containing 0.1% Tween 20, membranes were incubated with secondary antibody for anti-rabbit or anti-mouse IgG horseradish peroxidase-conjugated (1:3000, Cell Signaling) for one hour at room temperature. Bands of target proteins were detected using an ECL detection system (Wako). ImageJ Fiji (NIH) was used to analyses the bands. Data are showed as the mean ± standard error of the mean (SEM). GAPDH was used as the loading control.