Western blot was performed using Anti-CTCF Polyclonal Antibody (Product # PA5-88115) and a 110 kDa band corresponding to CTCF was observed across samples tested. The expression was reduced in MCF7 upon Trichostatin A treatment. Nuclear enriched extracts (30 µg lysate) of HeLa (Lane 1), HCT 116 (Lane 2), Hep G2 (Lane 3), LNCaP (Lane 4), PC-3 (Lane 5), HEK-293 (Lane 6), Mouse Thymus (Lane 7) in Fig. a; MCF7 (Lane 1), MCF7 treated with Trichostatin A (10 µM for 24 hrs) (Lane 2) in Fig.b 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 # A27036,1:20000 dilution using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Product # 34580). Additional uncharacterized bands at ~90, ~50 kDa were observed.

Immunofluorescence analysis of CTCF was performed using 70% confluent log phase MCF7 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 CTCF Polyclonal Antibody (Product # PA5-88115) 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 nuclear localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Immunofluorescence analysis of CTCF in NIH/3T3 cells. Samples were incubated with CTCF Polyclonal antibody (Product # PA5-88115) using a dilution of 1:50 (40x lens). Blue: DAPI for nuclear staining.