Immunofluorescence analysis of SMC3 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 SMC3 Polyclonal Antibody (Product # PA5-29131) at 5 µg/mL in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 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.

Western blot was performed using Anti-SMC3 Polyclonal Antibody (Product # PA5-29052) and a 160 kDa band corresponding to SMC3 was observed in all the tested cell and tissue models. Modified whole cell lysate (1% SDS) (30 µg lysate) of A-431 (Lane 1), HeLa (Lane 2), MCF7 (Lane 3), A549 (Lane 4), U-87 MG (Lane 5), SH-SY5Y (Lane 6), HCT 116 (Lane 7), Jurkat (Lane 8) and tissue extract (30 µg lysate) of Mouse Liver (Lane 9) were electrophoresed using NuPAGE® 10 % Bis-Tris gel (Product # NP0302BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # LC2001) by wet transfer. The blot was probed with the primary antibody (0.5 µg/mL) and detected by chemiluminescence with Goat anti-Rabbit IgG (Heavy Chain), Superclonal™ Recombinant Secondary Antibody, HRP conjugate (Product # A27036, 1:4,000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076).