Chemidoc xrs docking system

Cells were lysed by either mammalian protein extraction reagent or radio-immunoprecipitation assay (RIPA) buffer (Pierce Biotechnology, Rockford, IL, USA). Protein concentrations were determined using Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA). Equal amount of protein samples (60–100 µg) were separated by 8–12% SDS-PAGE and separated proteins were transferred onto polyvinylidene fluoride (PVDF) membrane for overnight. The transferred protein membranes were blocked with 5% non-fat skim milk for 30 min, followed by incubation with specific primary antibodies for overnight and either horseradish peroxidase-conjugated anti-rabbit or anti-mouse or anti-goat antibodies for 2 h. Immunoblots were developed with the enhanced chemiluminescence (ECL) reagents (Advansta Inc., San Jose, CA, USA), images were captured by ChemiDoc XRS+ docking system and the protein bands were quantified by using Imagelab software (Bio-Rad laboratories, Hercules, CA, USA).

B16-F10 cells were seeded in to 10 cm cell culture dishes with a density of 1 × 10⁶ cell/dish. After an overnight incubation, cells were treated with melanogenesis inducer (a mixture of 20 μM FSK and 100 nM α-MSH) or various doses of test samples for 48 h. Cells were lysed by radio-immuno precipitation assay (RIPA) buffer (Pierce Biotechnology, Rockford, IL, USA). Protein concentration in the lysates were determined by Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA). In total, 100 µg/lane of protein samples were separated by 10% SDS-PAGE. Then, the separated proteins were transferred onto polyvinylidene fluoride (PVDF) membrane for overnight. After the transfer, PVDF membranes were blocked with 5% skimmed milk for 90 min. After washed the membranes with TBST, the membranes were incubated with TYR, TRP-1, TRP-2, MITF, β-actin or GAPDH antibodies for overnight or 16 h, and then incubated with either HRP-conjugated anti-rabbit or anti-mouse antibodies for 2 h. Immunoblots were developed with the enhanced chemi-luminescence (ECL) reagents (Advansta Inc., San Jose, CA, USA), images were captured by ChemiDoc XRS+ docking system (Bio-Rad laboratories), and the protein bands were quantified by using Imagelab software (Bio-Rad laboratories).

RAW264.7 cells (1 × 10⁶ cells/dish in 6-cm dish) were co-treated with LPS with or without various doses of GTEO (25–100 μg/mL) for various time points. After treatment, cells were detached and washed in cold PBS twice. Then, they were lysed in a radioimmunoprecipitation assay (RIPA) buffer (Pierce Biotechnology, Rockford, IL, USA). The concentrations of proteins were determined using a Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein samples (60–100 mg/well) along with sample dye were denatured for 5 min at 94 °C. SDS-PAGE was used to separate the protein samples, followed by overnight transfer onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 0.1% Tween-20 in PBS containing 5% non-fat skim milk for 30 min at room temperature, reacted with primary antibodies for 2 h, and then incubated with either HRP-conjugated goat anti-rabbit or anti-mouse antibodies for 1 h. An enhanced chemiluminescence reagent (Advansta, Inc., San Jose, CA, USA) was used to develop the immunoblots, images were captured by the ChemiDoc XRS⁺ docking system, and the protein bands were quantified by using Imagelab software (Bio-Rad Laboratories, Hercules, CA, USA).