Anti-Human FOXM1 monoclonal antibody

For immunofluorescence analysis, HCT 116 cells were fixed and permeabilized for detection of endogenous FOXM1 using Anti- FOXM1 recombinant rabbit monoclonal antibody and labeled with Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor® 488 conjugate. Panel a) shows representative cells that were stained for detection and localization of FOXM1 protein (green), Panel b) is stained for nuclei (blue) using Gold Antifade Mountant with DAPI. Panel c) represents cytoskeletal F-actin staining using Rhodamine Phalloidin. 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.

Western blot analysis was performed on Nuclear extracts (30 µg lysate) of MCF-7 (Lane 1), NTERA-2 cl.D1 (Lane 2), U-2 OS (Lane 3), A549 (Lane 4) and A-431 (Lane 5). The blots were probed with Anti-FOXM1 Rabbit Monoclonal Antibody. A 84 kDa band corresponding to FOXM1 was observed across the cell lines tested. The blots were detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP conjugate.

Flow Cytometry analysis of endogenous FOXM1 was performed on U-2 OS cells. Cells were either untreated (asynchronous) or treated with 100 ng/mL nocodazole for 16 hours to arrest cells in the G2/M phase of the cell cycle, where FOXM1 levels are known to be highest. Cells were then released from arrest with medium containing 10% FBS for 3 and 18 hours. FOXM1 was detected using Anti-FOXM1 recombinant rabbit monoclonal antibody followed by Goat anti-Rabbit IgG (H+L) Secondary Antibody, (Alexa Fluor® 488 conjugate, , 0.4 µg/ml, 1:2500). Maximal expression of FOXM1 (panels a-d) was found to correlate with cells arrested in the G2/M phase as observed by staining with propidium iodide (panels e-h). Rabbit IgG was used as the isotype control to determine nonspecific binding.