Bicinchoninic acid protein assay kit

Total protein was extracted from H1299 and A549 cells with a radioimmunoprecipitation assay buffer with 0.5% SDS and 3% proteinase inhibitor cocktail (Sigma-Aldrich; Merck KGaA) for 30 min on ice. The concentration of protein was determined using the bicinchoninic acid protein assay kit (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). For western blotting, proteins were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Invitrogen; Thermo Fisher Scientific, Inc.). Subsequent to blocking with 5% bovine serum albumin (Gibco; Thermo Fisher Scientific, Inc.) at room temperature for 1 h, membranes were incubated with primary antibody against CDK9 (cat no. ab76320; dilution, 1:200; Abcam, Shanghai, China) at 4°C overnight. Membranes were then incubated with secondary antibody goat anti-rabbit IgG (cat no. sc-2007; dilution, 1:2,500; Santa Cruz Biotechnology, Inc.) for 2 h at room temperature. The signals were detected using a RapidStep^™ ECL Reagent (EMD Millipore, Billerica, MA, USA).

Total protein was extracted using a radioimmunoprecipitation assay lysis buffer (Sigma-Aldrich; Merck KGaA) and quantified using a bicinchoninic acid protein assay kit (Santa Cruz Biotechnology, Inc.). The samples (100 µg) were separated by 10% SDS-PAGE, transferred onto a polyvinylidene difluoride membrane, and then blocked for 1 h at room temperature by using of 5% bovine serum albumin (MP Biomedicals, LLC). The membranes were incubated with primary antibodies at 4°C overnight and subsequently with secondary antibodies [goat anti-rabbit immunoglobulin G horseradish peroxidase (HRP)-conjugated; cat. no. ab205718; dilution, 1:2,000; Abcam) at room temperature for 1 h. The primary antibodies used in the present study were the following: Anti-MMP-13 (cat. no. ab39012; 1:3,000 dilution), anti-ADAMTS5 (cat. no. ab231595; 1:250 dilution) and anti-GAPDH (cat. no. ab9485; 1:500 dilution; all Abcam). Following washing with 0.1% TBST 3 times, an ECL Western Blotting Substrate kit (cat. no. ab65623; Abcam) was used for chemiluminescence imaging and bands were analyzed with ImageJ software version 2 (National Institutes of Health).

The sample was sequentially centrifuged for 10 minutes at 14,000 × g, and then centrifuged for 15 minutes at 12,000 × g. After the supernatant was taken, an additional centrifugation for 30 minutes at 12,000 × g was performed. Lysate and protease inhibitor were added, and protein concentration was measured with bicinchoninic acid protein assay kit (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The sample was mixed with buffer solution, boiled for denaturation, and separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein was transferred to nitrocellulose membrane. The membrane was blocked with 5% skim milk, and incubated with primary antibodies, including rat anti-mouse Fyn monoclonal antibody (1:1,000; Stressgen, Victoria, Canada), rat anti-NR2B polyclonal antibody (1:1,000; Stressgen), rat anti-phospho-NR2B polyclonal antibody (1:1,000, Thermo Fisher Scientific Inc., St. Louis, MO, USA) and β-actin (1:1,000; Abcam, Cambridge, UK). The proteins were detected using horseradish peroxidase conjugated goat anti-rat secondary antibodies (1:1,000; Abcam) and visualized using chemiluminescence reagents provided with the enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The intensity of blots was quantified using NIH image J software (NIH, Bethesda, MD, USA).

Total protein of tissue specimens and cells were lysed by ice-cold radio immunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, Merck KGaA). Following protein concentration quantification with a Bicinchoninic Acid protein assay kit (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), 40 µg protein samples were subjected to 10% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (PVDF; Amresco, LLC, Solon, OH, USA) and then blocked by 5% BSA (Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h at room temperature. Subsequently, membranes were incubated with antibodies against SMAD4 (1:5,000; cat no. ab40759; Abcam, Cambridge, MA, UK), E-cadherin (1:100; cat. no. ab76055; Abcam), fibronectin (1 µg/ml; cat. no: ab23750; Abcam) and GAPDH (1:10,000; cat. no. ab128915; Abcam) at 4°C overnight. The following day, the membranes were incubated with secondary antibodies [goat anti-rabbit IgG horseradish peroxidase (HRP), 1:2,000; cat. no. ab205718, Abcam] at room temperature for 1 h. Following washing with TBST 3 times, an ECL Western Blotting Substrate kit (cat. no. ab65623; Abcam) was used for chemiluminescence imaging with Image J software version 2X (National Institutes of Health, Bethesda, MD, USA).

The cerebral cortex from six rats in each group was lysed in 1 mL of ice-cold radioimmunoprecipitation assay buffer containing protease inhibitors (Santa Cruz Biotechnology Inc., Dallas, TX, USA). Total protein was quantified using a bicinchoninic acid protein assay kit (Santa Cruz Biotechnology Inc.). An equal volume of protein underwent sodium dodecyl sulphate-polyacrylamide gel electrophoresis and was then transferred onto polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). Membranes were blocked with 5% skimmed milk, incubated with rabbit BKCa-α (1:500; Santa Cruz Biotechnology Inc.) and β-actin antibody (1:1,000; Santa Cruz Biotechnology Inc.) at 4°C overnight, and then horseradish peroxidase-labeled goat anti-rabbit IgG (1:1,000; Santa Cruz Biotechnology Inc.) at room temperature for 2 hours, followed by enhanced chemiluminescence (Thermo Fisher Scientific Inc., Waltham, MA, USA). Membranes were visualized and photographed using a gel imaging system (Bio-Rad, Hercules, CA, USA). Optical density values were determined using Quantity One image analyzer software (Bio-Rad). β-Actin served as the internal reference.