Immunofluorescence analysis of GTF2B was performed using 70% confluent log phase HeLa 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 1 hour at room temperature. The cells were labeled with GTF2B Polyclonal Antibody (Product # PA5-28556) at 1:100 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32731) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing 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-GTF2B Polyclonal Antibody (Product # PA5-28556) and 36 kDa band corresponding to GTF2B was observed across the cell lines and tissues tested. An uncharacterized band of ~ 110 kDa was also observed in all the cell lines. Modified whole cell extracts (1% SDS) (30 µg lysate) of HeLa (Lane1), K-562 (Lane 2), MOLT-4 (Lane 3), Hep G2 (Lane 4), Jurkat (Lane 5), MCF7 (Lane 6), tissue extracts (30 µg lysate) of Mouse Lungs (Lane 7) and Mouse Spleen (Lane 8) were electrophoresed using Novex® NuPAGE™ 10% Bis-Tris Protein Gel (Product # NP0302BOX). 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).