Amersham ecl plus western blotting detection kit

SW1353 cells (1.2 × 10⁶ per dish) were cultured overnight, and then added PKE (60 μg/mL), PKE (60 μg/mL) with 3.6 mM added H₂O₂, and 3.6 mM H₂O₂ were treated for 24 h. After the medium was removed, the total cell protein was extracted by lysing the cells in a buffer containing 50 mM Tris-Cl, 150 mM NaCl, 0.1% Triton X-100, 0.1% (m/v) SDS, 1.0 mM sodium orthovanadate, and 1 mM NaF at pH 8.0. Protein concentrations were determined by Bradford protein assay at an absorbance of 595 nm. Equal amounts of proteins were subjected to 10% SDS-polyacrylamide gel (SDS-PAGE), electrically transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA), blocked with blocking buffer (5% skimmed milk powder in PBS), and then probed with primary antibodies (PARP; 1:1000; Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. Blots were washed and incubated for 1 h with IRDye800^®-conjugated secondary antibodies (1:8000; Cell Signaling Technology, USA), and then washed with 0.1% Tween-20 in Tris-buffered saline (TBS). The proteins were visualized via chemiluminescence using the Amersham ECL Plus Western Blotting Detection kit (GE Healthcare, Chicago, IL, USA) according to the manufacturer’s instructions. β-Actin (Cell Signaling Technology, USA) was used as the loading control for protein expression in the treated cells.

PDLSCs were treated with 500 μg/mL DAE for seven days, and total cell protein was extracted by lysing the cells in radioimmunoprecipitation assay buffer (RIPA buffer). Protein concentrations were determined using Bradford protein assays with spectrophotometry at an absorbance of 595 nm. Samples (20 mg total protein) were subjected to SDS–polyacrylamide gel electrophoresis (SDS-PAGE) in a 10% gel, electrically transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) using 80 volts for 100 min, blocked with blocking buffer (5% skimmed milk powder in tris-buffered saline [TBS] containing 0.1% [v/v] Tween-20) for 1 h, and then probed with the primary antibody (OCN, 1:500; Abcam, Cambridge, UK) overnight at 4 °C. Blots were washed and placed on an orbital shaker for 1 h with secondary antibodies (IRDye800-conjugated, 1:10,000; Abcam), and then washed with 0.1% Tween-20 in TBS. The proteins were visualized via chemiluminescence (LAS-4000; Fujifilm, Minato, Japan) using the Amersham ECL Plus Western Blotting Detection kit (GE Healthcare, Chicago, IL, USA) according to the manufacturer’s instructions. β-actin was used as a loading control for protein expression in the treated cells.

MCF-7 cells were seeded in 6-well plates and exposed to the indicated concentrations of ginsenoside Rg1 with or without PMA. After treatment, the cells were harvested using lysis buffer (pH 7.4, 20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM Na₃VO₄, 1 mM β-glycerophosphate, 1:1,000 protease inhibitors). Protein concentrations were determined by the BCA method. Total protein (25 μg) was then separated using 8–12% sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose blotting membranes. The membranes were probed with monoclonal antibodies against MMP-9, MMP-2, TIMP-1 or β-actin (1:8,000), and immunopositive bands were visualized using the Amersham ECL™ Plus Western Blotting Detection kit (GE Healthcare, Piscataway, NJ, USA).

Whole-cell lysates were prepared in lysis buffer (20 mM Tris-HCl [pH7.4], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM Na₃VO₄, 1 mM β-glycerophosphate, and 1:1,000 protease inhibitors), sonicated and centrifuged at 14,167 g for 30 min at 4 °C. Protein concentration was determined by using a DC protein assay kit (Bio-Rad Co., Hercules, CA, USA). In total, 25 μg of protein from each sample was loaded on a SDS–PAGE, separated by electrophoresis, and transferred to a nitrocellulose membrane. Membranes were blocked at room temperature for 1 h, followed by incubation with specific antibodies overnight at 4 °C. Monoclonal antibodies for Cx32 (1:1,000), Cx43 (1:3,000), CTR1 (1:1,000), MRP2 (1:100) and β-actin (1:10,000) were used. Immuno-positive bands were visualized using an Amersham ECL™ Plus Western Blotting Detection Kit (GE Healthcare, Piscataway, NJ, USA).

AllPrep^® DNA/RNA/Protein Mini Kit (QIAGEN) was used to extract total protein from MG6. After measuring protein concentrations by BSA protein Assay Kit (Thermo Fisher Scientific, Cleveland, OH, USA), the same levels of protein from each sample were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad) and western blotting with the following primary antibodies: polyclonal rabbit anti-TNFα antibody (1:2000; Abcam, ab6671, Cambridge, UK) and monoclonal mouse anti-ACTB antibody (1:1000; Sigma-Aldrich). The secondary antibodies employed were horseradish peroxidase-conjugated anti-rabbit IgG (1:2000; Dako, Glostrup, Denmark) and anti-mouse IgG (1:3000; Jackson ImmunoResearch, West Grove, PA, USA), respectively. Chemiluminescence was detected using an Amersham ECL Plus western blotting detection kit (GE Healthcare, Waukesha, WI, USA) and a ChemiDoc MP Imaging System (Bio-Rad), and the results were quantified using ImageJ 13.0.6 software (http://rsb.info.nih.gov/ij/, accessed on 3 February 2023) [65 (link)].

Cells were harvested using lysis buffer (Tris•HCl pH 7.4 20 mM, NaCl 150 mM, EDTA 1 mM, EGTA 1 mM, Triton 1%, sodium pyrophosphate 2.5 mM, Na₃VO₄ 1 mM, β-glycerophosphate 1 mM, protease inhibitors 1:1000). Monoclonal antibodies against Cx43 (Sigma, C8093, 1:5000) or α-tubulin (Sigma, T9026, 1:10000) were used. Immunopositive bands were visualized by Amersham ECL™ Plus Western Blotting Detection Kit (GE Healthcare), and were quantitatively analyzed using image J software.