Gel doc chemi doc universal hood 2

The cells were washed 3 times in ice-cold PBS and lysed in RIPA buffer (pH 7.4, 50 mM tris, 0.1% SDS, 50 mM NaCl, 1% NP-40, 1 mM PMSF, 10 μg/mL aprotinin and 10 μg/mL leupeptin). An aliquot of the lysate was used to determine the protein content by the method of Bradford assay using bovine serum albumins as the standard. The proteins (40 μg) were separated on 10% SDS-polyacrylamide gel electrophoresis and blotted onto Hybond-C Extra nitrocellulose membrane (Amersham Bioscience, U.K.). The membranes were blocked with 5% non-fat milk in Tris-buffer saline (TBS) containing 10 mM Tris-HCl, pH 7.5 and 150 mM NaCl for 30 min. MITF, tyrosinase, dopachrome tautomerase 2 (TRP-2), TRP-1 and β-actin (as an internal control) were detected using a rabbit polyclonal antibodies, respectively. The membranes were further incubated with goat polyclonal secondary antibody to rabbit IgG-H&L (HRP). All bound antibodies were then detected using Super Signal® West Pico Chemiluminescent Substrate (ECL) (Thermo Scientific). The signal intensity of each band was quantified with a densitometer system Gel Doc TM / Chemi Doc TM Universal hood II (Bio-Rad) equipped with an integrator, and normalized with that of the internal control.

The NF-κB production in the cell were determined by western blot as described previously [8 (link)]. The cells were lysed in RIPA buffer (pH 7.4, 50 mM tris, 0.1% SDS, 50 mM NaCl, 1% NP-40, 1 mM PMSF, 10 μg/mL aprotinin and 10 μg/mL leupeptin) and collected its nuclear fraction. Protein quantified by Bradford assay using BSA as the standard and the proteins were separated by 10% SDS-PAGE, transferred onto hybond-C Extra nitrocellulose membrane (Amersham Biotscience, U.K.). The membranes were blocked overnight in block solution (TBS-T buffer containing 5% non-fat skim milk). The membrane was then incubated with rabbit polyclonal anti-NF-κB and antiβ-actin antibodies as an internal control. The membranes were further incubated with HRP conjugated anti-rabbit goat polyclonal secondary antibody. Proteins were detected by chemilluminescent with Super Signal® West Pico Chemiluminescent Substrate (ECL) (Thermo Scientific). The signal density of each band was measure and analyzed using a densitometer system Gel Doc TM / Chemi Doc TM Universal hood II (Bio-Rad).