Anti mitf

After 48 h transfection, cells were trypsinized and washed three times with PBS and then lysed in lysis buffer for 30 min on ice. The protein was then electrophoresed on an AnyKD SDS-PAGE gel (Bio-Rad, Hercules, CA, USA) and then transferred to a nitrocellulose membrane (Bio-Rad) and blocked with 5% non-fat milk in TBST for 2 h at room temperature with shaking. The membrane was immunoblotted with primary antibody for rabbit anti-GAPDH, anti-MITF and anti-MAP4K3 (Cell Signaling Technology, Danvers, MA, USA) with dilutions of 1 : 1000, 1 : 1000 and 1 : 1000, respectively, at 4 °C overnight. Signals were developed using HRP-linked secondary antibody (1 : 10 000) with Clarity Western ECL Substrate (Bio-Rad). The intensity of the signals was determined by FluorChem E system (Protein Simple, Santa Clara, CA, USA).

Penicillin/streptomycin (P/S), trypsin–ethylenediamine tetra-acetic acid (EDTA), and FBS were purchased from Gibco BRL (Gaithersburg, MD, USA) and Dulbecco’s modified eagle medium (DMEM) and phosphate-buffered saline (PBS) from Welgene (Daegu, Korea). Triton X-100, L-DOPA, bovine serum albumin (BSA), α-MSH, phenylmethylsulfonyl fluoride, NaOH, 10× DMEM, Tween-20 and dimethyl sulfoxide (DMSO) were from MilliporeSigma (St. Louis, MO, USA). Recombinant human epidermal growth factor (EGF: purity > 97%) was obtained from R&D Systems (Minneapolis, MN, USA). EZ-CyTox kits were supplied by DoGenBio (Seoul, Korea) and type I collagen by BD Bioscience (Franklin Lakes, NJ, USA). The antibodies used were as follows: anti-ERK1/2, anti-phospho ERK1/2, anti-AKT, anti-phospho AKT, anti-JNK, anti-phospho JNK, anti-p38 MAPK, anti-phospho p38 MAPK, anti-rabbit IgG, anti-MITF, and anti-mouse IgG (all from Cell Signaling, Beverly, MA, USA); polyclonal anti-type I, and IV collagen, monoclonal anti-type I and IV collagen (Abcam; Cambridge, UK); β-actin (MilliporeSigma); and anti-tyrosinase (Santa Cruz Biotechnology, CA, USA).

Western blot analysis has been described previously^{42 (link)}. Antibodies used in this study were: anti-MITF (12590 S, Cell Signaling Technologies [CST], Danvers, MA), anti-BRN2 antibody (12137 S, CST), anti-MLANA antibody (M7196, Dako, Santa Clara, CA), anti-PAX3 antibody (701147, Invitrogen), anti-phospho-STAT3 antibody (Y705; 91455, CST), anti-STAT3 antibody (9139 S, CST), anti-c-MET antibody (D1C2; 8198 S, CST), anti-b-galactosidase antibody (2372 S, CST), anti-TWIST1 antibody (46702 S, CST), anti-β1-integrin antibody (34971, CST), and anti-GAPDH (R&D Systems, Minneapolis, MN).

Western Blot analysis was performed as described previously [14 (link)]. The following primary antibodies were used: anti-ERK, anti-phospho-ERK (Thr202/Tyr204), anti-AKT, anti-phospho- AKT (Thr473), anti-Mcl-1, anti-beta-actin, anti-phospho-eIF2a, anti-eIF2a, anti-LC3, anti-Atg5, anti-MITF (Cell Signaling Technology). UACC257 cells were cultured in Vitamin D deficient medium for more than one week, and then treated with different concentration of α-Mangostin (5 μM or 0.5 μM), ATRA (5 μM or 0.5 μM) or their combination for 8 hours. The expression level of MITF was detected by western blot. ChemiDoc XRS system (Bio-Rad) was used for imaging.

Tissue microarrays (TMAs) containing 46 cases with paired tumor and non-tumor tissues and 13 cases without corresponding non-tumor tissues were analyzed in this study. TMA was obtained from Xinchao Biotech, Shanghai, China. Paraffin tissue sections were dewaxed and rehydrated. Antigen retrieval was performed by heating the slides in sodium citrate buffer (10 mM, pH 6.0). After blocking with bovine serum albumin (Sango Biotech, Shanghai, China), the slides were incubated with anti-c-Met (Epitomics, Burlingame, CA, USA), anti-MITF (Cell Signaling Technology, Beverly, MA, USA), or anti-CREB1 (Cell Signaling Technology) overnight at 4 °C. The slides were then incubated with a secondary antibody of goat anti-rabbit HRP conjugate for 1 h at room temperature. A DAB solution was used for brown color development. The strength of positivity was semi-quantified by considering both the intensity and proportion of positive cells.

Melan-a cell lysates were prepared using a standard protocol, mixed with 5X SDS-PAGE (3M Science, Seoul, Korea) sample buffer and denatured at 100 °C for 5 min. An equal amount (20 µg protein) of each sample was separated by 10% SDS-PAGE gel electrophoresis, followed by electrotransfer to nitrocellulose membranes (Whatman, Dassel, Germany). The membranes were then incubated overnight with 5% skim milk (for anti-Tyr, anti-TYRP-1, anti-TYRP-2, anti-MITF) or 5% bovine serum albumin (BSA) for anti-phospho-JNK, anti-JNK, anti-phospho-p38, anti-p38, and Anti-p44/42MAPK (ERK1/2), and anti-phospho-p44/42MAPK (ERK1/2), detection. anti-Tyr, anti-TYRP-1, anti-TYRP-2, anti-MITF (Bioworld Technology, St. Louis Park, MN, USA) for Western blotting, and anti-phospho-JNK, anti-JNK, anti-phospho-p38, anti-p38, and Anti-p44/42MAPK (ERK1/2), and anti-phospho-p44/42MAPK (ERK1/2), and anti-MITF (Cell Signaling Technology, Beverly, MA, USA) for signaling studies were utilized as 1st antibodies. Anti-goat IgG-horse radish peroxidase (HRP) and anti-mouse IgG-HRP were purchased from Santa Cruz and used as 2nd antibodies. The reaction was proceeded using an ECL system (Perkin Elmer).