Rabbit anti phospho h2a x ser139

Manufactured by Cell Signaling Technology

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Rabbit anti-phospho-H2A.X (Ser139) is a primary antibody that specifically recognizes the phosphorylated form of the histone variant H2A.X at serine 139. This antibody can be used to detect and quantify DNA damage response signaling.

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Close spelling variants of this product

Rabbit anti-phospho-Histone H2A.X (ser139) (10 citations) Anti-H2A (9 citations) Rabbit anti-Phospho-Histone H2A.X(Ser139) antibody (4 citations) Anti-phospho-Ser139 H2A.X (2 citations)

4 protocols using rabbit anti phospho h2a x ser139

Western Blotting and Cell Fractionation

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Western blotting was performed as already described^{55 (link)}. The following antibodies were used: mouse anti-GLI1 (#2643), mouse anti-cyclin A2 (#4656), rabbit anti-BCL2 (#2876), rabbit anti-BAX (#2772), rabbit anti-cyclin B1 (#12231), rabbit anti-PARP-1 (#9532), rabbit anti-phospho-ATR (Ser428) (#2853), rabbit anti-phospho-CHK1 (Ser345) (#2348), rabbit anti-phospho-CDC2 (Tyr15) (#4539), rabbit anti-phospho-H2A.X (Ser139) (#9718), rabbit anti-phospho-Histone H3 (Ser10) (#3377), rabbit anti-phospho-WEE1 (Ser642) (#4910) (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-CDC2 (sc-954), mouse anti-Myc (sc-40), mouse anti-HSP90 (sc-13119) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit anti-SMO (ST1718) (Merck Millipore, Burlington, MA, USA). Chemiluminescent detection was used. Cell fractionation was performed as previously described^{56 (link)}. The following antibodies were used: mouse anti-GLI1 (#2643) (Cell Signaling Technology), goat anti-fibrillarin (D-14), and goat anti-GAPDH (V-18) (Santa Cruz Biotechnology).

Pietrobono S., Santini R., Gagliardi S., Dapporto F., Colecchia D., Chiariello M., Leone C., Valoti M., Manetti F., Petricci E., Taddei M, & Stecca B. (2018). Targeted inhibition of Hedgehog-GLI signaling by novel acylguanidine derivatives inhibits melanoma cell growth by inducing replication stress and mitotic catastrophe. Cell Death & Disease, 9(2), 142.

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Western Blot Analysis of Protein Modifications

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Dissected brain tissues and MEFs were lysed with ice-cold RIPA lysis buffer (ThermoFisher Scientific) containing Halt™ protease and phosphatase inhibitor cocktail (ThermoFisher Scientific). Lysed issue and cells samples were microcentrifuged for 15 min at 14,000 rpm and the supernatants were collected. Proteins were separated by SDS-PAGE (4–12% Bis-Tris gels) and transferred to PVDF membranes using a BioRad wet transfer system. Subsequently, membranes were blocked for 2 h in 5% non-fat dry milk and incubated overnight with rabbit anti-phospho-H2A.X (Ser139) (#2577, Cell Signaling) in tris-buffered saline with Tween 20 (TBST) or rabbit anti-4-hydroxynonenal (HNE) primary antibody (ab46545; Abcam) in phosphate-buffered saline with Tween 20 (PBST) containing 5% non-fat dry milk. Membranes were washed 3X with 1XTBST and 1X PBST respectively for 15 min and then incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories) for 1 h with constant rocking at room temperature. Signal was detected using enhanced chemiluminescence (Amersham) and quantified with NIH image J software.

Khan M.M., Xiao J., Patel D, & LeDoux M.S. (2018). DNA damage and neurodegenerative phenotypes in aged Ciz1 null mice. Neurobiology of aging, 62, 180-190.

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Western Blot Analysis of Protein Acetylation

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Cells or tissues were lysed with radio-immunoprecipitation assay buffer. Western blotting was carried out by standard protocols. The following primary antibodies were used: mouse anti-TSP-1 (Abcam, Cambridge, MA); rabbit anti-HDAC4 (Signalway Antibody, Pearland, TX); rabbit anti-GAPDH, rabbit anti-P21, rabbit anti-HDAC8 (Proteintech Group, Chicago, IL); rabbit anti-acetylated histone H3, rabbit anti-acetylated histone H3 (Lys14, 27, 56 and K18), rabbit anti-phospho-H2AX ser-139 (Cell Signaling Technology, Danvers, MA), synaptophysin (Abcam, Cambridge, MA, USA). Proteins were visualized with horseradish peroxidase (HRP)-conjugated anti-rabbit, anti-mouse IgG, (Cell Signaling Technology, Danvers, MA) followed by use of the ECL chemiluminescence system. Western blot data was subjected to densitometry analysis by computerized image analysis and software (Gel-Pro Analyzer software ver.6.0, Media Cyberetics, USA).

Chen P., Yu N., Zhang Z., Zhang P., Yang Y., Wu N., Xu L., Zhang J., Ge J., Yu K, & Zhuang J. (2016). Thrombospondin-1 might be a therapeutic target to suppress RB cells by regulating the DNA double-strand breaks repair. Oncotarget, 7(5), 6105-6120.

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Immunofluorescent Characterization of Cell Lines

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WERI-Rb1 cells, Y79 cells or tissue sections were fixed in methanol and then were characterized by staining with mouse anti-TSP-1 (Abcam, Cambridge, MA, USA) [54 (link)], synaptophysin (Abcam, Cambridge, MA, USA and rabbit anti-phospho-H2AX ser-139 (Cell Signaling Technology, Danvers, MA), respectively. Secondary anti-mouse antibodies (CST, Lenexa, KS, USA) were added at room temperature, and the nuclei were stained with DAPI. The cells were counterstained with the fluorescent nuclear-binding label 4,6-diamidino-2-phenylindole. Images were obtained by fluorescence microscopy.

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