Anti tyrp 1 antibody

The cells were lysed with 1% NP-40 in a solution of 0.05 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.01 M MgCl₂. The cell debris was removed by centrifugation at 16,000 ×g for 20 minutes. The supernatants were removed, and the protein content was quantified using a BCA assay. An aliquot of 20 µg of protein was loaded into each well of an SDS-PAGE gel. For immunoblotting, the SDS-PAGE gel was electroblotted onto a PVDF membrane. An anti-TYRP-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-GAPDH antibody (Santa Cruz Biotechnology), anti-MART-1 antibody (Thermo Fisher Scientific, CA, USA), anti-HMB45 antibody (GeneTex, Irvine, CA, USA), anti-TA99 antibody (GeneTex), anti-PMEL17 (aN) (GeneTex) and anti-TYR antibody (Upstate Biotechnology, Lake Placid, NY, USA) were used for protein detection. For the fluorescence microscopy, cells were cultured on Lab-Tek chamber slides (Nunc, NY, USA), fixed with 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 in PBS containing 1% BSA for 5 minutes, and incubated with a primary antibody. Fluorescence was detected by secondary antibody staining with the Alexa Fluor 488-conjugated F(ab')2 fragment of goat anti-mouse IgG (Invitrogen) and the Alexa Fluor 594-conjugated F(ab')2 fragment of goat anti-rabbit IgG (Invitrogen).

B16F10 (3 × 10⁴) cells, seeded onto a poly l-Lysin-coated 18-mm slide glass, were treated with 10 µM GIF-2209 for 24–48 h. Cells were fixed with 4% paraformaldehyde and 0.025% Triton-X 100 (Nacalai Tesque, Kyoto, Japan). After blocking, cells were incubated with the primary antibody (anti-TYR antibody as described in [8 (link)], anti-TYRP-1 antibody was from Santa Cruz Biotechnology, Dallas, TX, USA, and anti-CD63 antibody was from MBL) (1/5000), then the secondary antibody (goat anti-rabbit IgG H&L [Alexa Fluor^® 488] and goat anti-mouse IgG H&L [Alexa Fluor^® 594], Abcam, Pleasanton, CA, USA). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, ANASPEC INC., CA, USA). Finally, cells were placed on a glass plate. Cell Navigator^® Lysosome Staining Kit Red Fluorescence was acquired from ATT Bioquest (Sunnyvale, CA, USA), and the cells were stained according to the manufacturer’s protocol.