Immunofluorescence analysis of MiTF was performed using 70% confluent log phase A-375 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with MiTF Recombinant Rabbit Monoclonal Antibody (JF100-01) (Product # MA5-32554) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing predominantly nuclear localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60x magnification.

Western blot was performed using Anti-MiTF Recombinant Rabbit Monoclonal Antibody (JF100-01) (Product # MA5-32554) and a 55kDa band corresponding to MiTF was observed across all cell lines tested. Nuclear enriched extracts (30 µg lysate) of HeLa (Lane 1), PANC-1 (Lane 2), A-375 (Lane 3), SK-MEL-5 (Lane 4), SK-MEL-31 (Lane 5), B16-F10 (Lane 6), NIH/3T3 (Lane 7), MDA-MB-231 (Lane 8) and SK-BR-3 (Lane 9) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).