Western blot analysis of extracts from 293T cells, untreated (-) or UV-treated (50 mJ/cm2, 20 min recovery; +), using Phospho-ATF-2 (Thr69/71)/ATF-7 (Thr51/53) Antibody (upper), ATF-2/ATF-7 (A9G1M) Rabbit mAb #82870 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower)
Western blot analysis of extracts from HeLa cells, mock transfected (-) or transfected with a construct expressing Myc-tagged full-length human ATF-2 protein (hATF-2-Myc; +) or a construct expressing Myc-tagged full-length human ATF-7 protein (hATF-7-Myc; +), using Phospho-ATF-2 (Thr69/71)/ATF-7 (Thr51/53) Antibody (upper), Myc-Tag (71D10) Rabbit mAb #2278 (middle), and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Immunohistochemical analysis of paraffin-embedded human lung adenocarcinoma, untreated (left) or lambda phosphatase treated (right), using Phospho-ATF-2 (Thr69/71)/ATF-7 (Thr51/53) Antibody.
Immunohistochemical analysis of paraffin-embedded human non-small cell lung carcinoma using Phospho-ATF-2 (Thr69/71)/ATF-7 (Thr51/53) Antibody.
Immunohistochemical analysis of paraffin-embedded NIH/3T3 cell pellet, untreated (left) or treated with Anisomycin #2222 (right), using Phospho-ATF-2 (Thr69/71)/ATF-7 (Thr51/53) Antibody.
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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 BSA, 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.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
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 October 2016
Protocol Id: 10
NOTE: Do not allow slides to dry at any time during this procedure.
For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
posted June 2005
revised March 2016
Protocol Id: 310
Phospho-ATF-2 (Thr69/71)/ATF-7 (Thr51/53) Antibody detects endogenous levels of ATF-2 only when dually phosphorylated at both Thr69 and Thr71, and ATF-7 only when dually phosphorylated at both Thr51 and Thr53. It does not recognize ATF-2 singly phosphorylated at either Thr69 or Thr71, and it does not recognize ATF-7 singly phosphorylated at either Thr51 or Thr53.Species Reactivity:
Human, Mouse, Rat, Monkey
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr69 and Thr71 of human ATF-2. Antibodies are purified by protein A and peptide affinity chromatography.
The transcription factor ATF-2 (also called CRE-BP1) binds to both AP-1 and CRE DNA response elements and is a member of the ATF/CREB family of leucine zipper proteins (1). ATF-2 interacts with a variety of viral oncoproteins and cellular tumor suppressors and is a target of the SAPK/JNK and p38 MAP kinase signaling pathways (2-4). Various forms of cellular stress, including genotoxic agents, inflammatory cytokines, and UV irradiation, stimulate the transcriptional activity of ATF-2. Cellular stress activates ATF-2 by phosphorylation of Thr69 and Thr71 (2-4). Both SAPK and p38 MAPK have been shown to phosphorylate ATF-2 at these sites in vitro and in cells transfected with ATF-2. Mutations of these sites result in the loss of stress-induced transcription by ATF-2 (2-4). In addition, mutations at these sites reduce the ability of E1A and Rb to stimulate gene expression via ATF-2 (2).
ATF-7 is another member of the ATF/CREB family of leucine zipper proteins (5). Similarly, Thr51 and Thr53 (corresponding to Thr69 and Thr71 of ATF-2, respectively) can be phosphorylated under different conditions (6, 7).
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