Dye binding protein assay kit

Melan-a cells (1.5 × 10⁴ cells/ml) were cultured in a 60 mm dish for 24 h. And then, DGD was treated with or without indicated concentration and time. The cell were lysed in cold lysis buffer (20 mM Tris-HCL (PH 7.5), 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM glycerophosphate, 1 μg/ml leupeptin, 1 mM PMSF and a protease inhibitor). The protein concentration was determined using a dye-binding protein assay kit (Bio-Rad Laboratories Inc.), as described in the manufacturer’s manual. A 20-40 μg lysate protein was separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to a polyvinylidene fluoride membrane (Millipore Crop., USA). After blotting, the membranes were blocked with 5% non-fat skim milk in a Tris buffered saline-T buffer at 4°C overnight and incubated for 2 h with the specific primary antibodies (1:1000). After hybridization with secondary antibodies (1:5000, Santa Cruz Biotech, USA), the protein bands were visualized using an ECL plus Western blotting detection system (Amersham™, USA).

For Western blot assay, cells (1.5 × 10⁶ total) were cultured in a 10 cm dish for 48 hrs, followed by starvation in serum-free DMEM for 24 hrs. Cells were then treated with DHGA-D (2.5 or 5 μM) for 1 hr and irradiated with UVB (0.05 J/cm²). The protein concentration was determined by using a dye-binding protein assay kit (Bio-Rad Laboratories) following instructions in the manufacturer's manual. Lysate protein was subjected to 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech). After transferring, the membranes were incubated with specific primary antibodies at 4°C overnight. Protein bands were visualized by using a chemiluminescence detection kit (Amersham Pharmacia Biotech) after hybridization with a horseradish peroxidase-conjugated secondary antibody.

Cells were rinsed, scraped off and lysed using RIPA buffer with a protease and phosphatase inhibitor cocktail (Sigma–Aldrich, St. Louis, MO, USA). After centrifugation of the lysate, the supernatants were collected and quantified using a dye-binding protein assay kit (Bio-Rad, Hercules, CA, USA) or the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Proteins were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). After blocking in 5% skim milk in TBS, containing 0.1% Tween 20 (TBST), corresponding antibodies were applied to membranes and incubated overnight at 4 °C. After washing with TBST, a HRP-conjugated secondary antibody was applied to the membranes and bands were visualized using Western lightning Plus-ECL (PerkinElmer, Waltham, MA, USA).

For protein extraction, pooled mesenteric arteries were lysed in a buffer containing 150 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 8.5), 2 mmol/L EDTA, 1% v/v NP-40, 0.5% w/v deoxycholate, 10 mmol/L NaF, 10 mM sodium pyrophosphate, 2 mmol/L PMSF, 2 g/mL leupeptin, and 2 g/mL aprotinin, pH 7.4. Lysates were incubated on ice for 15 min and then centrifuged at 38,000× g for 30 min at 4 °C to collect the supernatant. Protein concentration was measured using a dye-binding protein assay kit (Bio-Rad) and reading to the spectrophotometer at a wavelength of 595 nm. Immunoblotting was performed as previously described [25 (link)], using the following antibodies: anti-bactin (Abcam, ab49900; mouse monoclonal, 1:4000), anti-phospho-ERK1/2 (Santa Cruz, sc-136521; mouse monoclonal 1:800), anti-AT1 receptor (Santa Cruz, sc-57036; mouse monoclonal 1:1000), anti-Rac1-GTPγ (STA-401-1, Cells Biolab Inc., 1:800). Secondary antibodies (1:3000) were purchased from Amersham Life Sciences (GE Healthcare). Bands were visualized with enhanced chemiluminescence (ECL, Amersham Life Sciences), according to the manufacturer’s instructions. Immunoblotting data were analysed using ImageJ software (developed by Wayne Rasband, NIH, Bethesda, MD, USA) to determine the density of the bands.

B16F10 cells (1.5 × 10⁴) were cultured in a 6 cm dish for 48 h then starved in serum-free medium for an additional 24 h to eliminate the influence of FBS on kinase activation. HEMs (9.6 × 10⁴) were cultured in 9 cm dish for 6 days. The culture media was replaced with the media containing 7,3′,4′-THIF (10, 20, or 40 μM) for 1 h before being exposed to 100 nM α-MSH for 3 days. The harvested cells were disrupted and the supernatant fractions were boiled for 5 min. The protein concentration was determined using a dye-binding protein assay kit (Bio-Rad Laboratories Inc.), as described in the manufacturer’s manual. Lysate protein (20–40 μg) was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to a polyvinylidene fluoride membrane (Millipore). After blotting, the membrane was incubated with primary antibodies at 4°C overnight. After hybridization with secondary antibodies, the protein bands were visualized using an ECL plus Western blotting detection system (Amersham^TM, Piscataway, NJ, United States). The relative intensities were quantified by Image J program.