For immunofluorescence analysis, HCT 116 cells were fixed and permeabilized for detection of endogenous FOXM1 using Anti- FOXM1 Recombinant Rabbit Polyclonal Antibody (Product # 711695, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Panel a) shows representative cells that were stained for detection and localization of FOXM1 protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of panels a, b and c clearly demonstrating nuclear localization of FOXM1. Panel e) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Western blot analysis was performed on whole cell extracts (30 µg lysate) of MCF-7 (Lane 1), NTERA-2 c1.D1 (Lane 2), A-431 (Lane 3) and A549 (Lane 4). The blots were probed with Anti-FOXM1 Recombinant Rabbit Polyclonal Antibody (Product # 711695, 1 µg/mL). A 100 kDa band corresponding to FOXM1 was observed across the cell lines tested. The blots were detected by chemiluminescence using Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:5000 dilution). Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).