Laemmli sample buffer

We purchased 2,2-diphenyl-1-picrylhydrazyl (DPPH), ascorbic acid, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), hydrogen peroxide, 2′,7′-dichlorofluorescin diacetate (DCFH-DA), and Dulbecco’s phosphate-buffered saline (DPBS) from Sigma-Aldrich Co., LLC (USA). Fetal bovine serum (FBS) was purchased from Gibco Thermo Scientific Inc. (USA). Hanks’ balanced salt solution (HBSS), Roswell Park Memorial Institute (RPMI)-1640, trypsin-EDTA, and penicillin-streptomycin solutions were purchased from Welgene Inc. (Republic of Korea). Laemmli sample buffer was obtained from FUJIFILM Wako Pure Chemical Corp. (Japan). RIPA Lysis and Extraction Buffer and Pierce bicinchoninic acid (BCA) Protein Assay Kit was purchased from Thermo Fisher Scientific Inc. Primary and secondary antibodies were obtained from Cell Signaling Technology (USA). All reagents and chemicals were of analytical or high-performance liquid chromatography (HPLC) grade unless otherwise specified.

To elucidate the phosphorylation status, we used phos-tag SDS-PAGE, which separates phosphorylated and non-phosphorylated proteins, based on their levels by capturing phosphate residues. Phos-tag gels were self-made using Phos-tag acrylamide reagent (Wako, Monza, Italy). Cell lysates or precipitates from co-immunoprecipitation assays were used for western blotting. Each sample was mixed with a Laemmli sample buffer (Wako, Monza, Italy), resolved on SDS-PAGE or phos-tag SDS-PAGE, and transferred to PVDF membranes. Protein-bound membranes were blocked with a 5% skim milk buffer and incubated with a primary antibody diluted in 0.5% skim milk buffer. Mouse anti-Myc antibody (1:2000, MBL) and mouse anti-GFP antibody (1:3000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used as primary antibodies. Horseradish peroxidase-conjugated anti-mouse IgG antibody was used as the secondary antibody (Agilent Technologies, Santa Clara, CA, USA). The membrane was subsequently washed and developed with ImmunoStar Zeta chemiluminescent reagents (Wako, Monza, Italy) and chemiluminescence was visualized using the Fusion (Vilber-Lourmat, Collégien, France).

Cells were washed three times with PBS containing 10 mM EDTA and then lysed using Laemmli sample buffer (Wako). Protein samples (5 or 10 μg) were subjected to SDS-polyacrylamide gel electrophoresis (4–20% gradient gels). Proteins were electrophoretically transferred to nitrocellulose membranes, blocked with PBS/0.1% Tween 20 (PBS-T) containing 5% nonfat dried milk, washed with PBS-T, and incubated with antibodies against Furin (1:1,000 dilution) in PBS-T containing 5% nonfat dried milk overnight. The anti-c-Myc antibody (1:200 dilutions) was diluted with PBS-T containing 2% bovine serum albumin. The membranes were washed three times with PBS-T and then incubated with a secondary antibody conjugated with horseradish peroxidase (1:5,000 dilutions) in PBS-T containing 5% nonfat dried milk for 1 h. Detection was performed using Supersignal West Dura Extended Duration Substrate (Thermo Fisher Scientific). Quantification of the chemiluminescent signals was performed with a digital imaging system (ChemiDoc, Bio-Rad)