Western blot analysis of extracts from U-2 OS cells synchronized in mitosis with Nocodazole #2190 (100 ng/ml, 18 hr) and released into fresh medium for the indicated times, using Phospho-FoxM1 (Ser35) Antibody (upper), FoxM1 (D12D5) XP® Rabbit mAb #5436 (middle) or α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower).
Western blot analysis of extracts from HeLa cells that are untreated (-), synchronized in S-phase by double thymidine block (2 mM, 18 hr; +), or synchronized in mitosis with double thymidine block followed by release into fresh medium containing Nocodazole #2190 (100 ng/ml, 18 hr; +), using Phospho-FoxM1 (Ser35) Antibody (upper), FoxM1 (D12D5) XP® Rabbit mAb #5436 (middle) or α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower).
Western blot analysis of extracts from ACHN cells that are untreated (-) or synchronized in mitosis with Nocodazole #2190 (100 ng/ml, 18 hr; +), using Phospho-FoxM1 (Ser35) Antibody (upper), FoxM1 (D12D5) XP® Rabbit mAb #5436 (middle) or α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower).
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised June 2016
Protocol Id: 263
Phospho-FoxM1 (Ser35) Antibody recognizes endogenous levels of FoxM1 protein only when phosphorylated at Ser35.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser35 of human FoxM1 protein. Antibodies are purified by protein A and peptide affinity chromatography.
Forkhead box M1 (FoxM1) is a forkhead box family transcription factor that regulates a number of genes throughout the cell cycle to help control DNA replication, mitosis, and cell proliferation. FoxM1 expression increases during G1 and S and reaches maximum levels in G2/M (1-3). Nuclear translocation occurs just before entry into G2/M and is associated with FoxM1 phosphorylation (4). Phosphorylation of FoxM1 by MAPK (Ser331, Ser704), Cyclin/Cdk (Ser4, Ser35, Thr600, Thr611, Thr620, Thr627, Ser638), Plk1 (Ser715, Ser724), and Chk2 (Ser376) stabilizes and activates FoxM1 (4-8). Forkhead box M1 is expressed in all embryonic tissues but is restricted to proliferating tissues in adults (9). Research studies show that FoxM1 expression is negatively regulated by p53 (10,11). Upregulation of FoxM1 is associated with many human cancers, including prostate, breast, lung, ovary, colon, pancreas, stomach, bladder, liver, and kidney, and may be associated with p53 mutations in some tumors (11,12). As a result, FoxM1 inhibitors have become a topic of interest for potential cancer therapy (13).
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