Cells were seeded in 6-well plates and allowed to attach for 24 h. Cells were then incubated for 24 hours with indicated concentrations of AZ31 and/or SN38. Cells were then washed with PBS and lysed with RIPA buffer (Cell Signaling, Danvers, MA). After sonication and centrifugation, a total of 30 μg of protein lysate was loaded onto a NuPage gel (Life Technologies, Carlsbad, CA), electrophoresed, and transferred to a nitrocellulose membrane using the Pierce G2 FastBlotter (Thermo Fisher, Rockford, IL). The membrane was blocked and probed overnight with primary antibodies (Cell Signaling Technologies (Danvers, MA) at a concentration 1:1000: p-ATM (catalog# 13050), ATM (catalog# 2873), p-p53 (catalog# 9284), p53 (catalog# 2527), p-CHK2 (catalog# 2665), p-RAD50 (catalog# 14223), p-H2AX (catalog# 9718) and actin (catalog# 4970). The next day the membranes were washed for 10 minutes 3X with TBS/Tween 20, and then probed with DyLight secondary antibodies 1:15,000 (Cell signaling, Danvers, MA), and imaged using the Licor Odyssey (Licor, Lincoln, NE).
P rad50
Manufactured by Cell Signaling Technology
Sourced in United States
P-RAD50 is a lab equipment product produced by Cell Signaling Technology. It is an antibody that specifically recognizes phosphorylated RAD50 protein.
Automatically generated - may contain errors
Lab products found in correlation
P chk2, by Cell Signaling Technology (1 mentions)
Pierce g2 fast blotter, by Thermo Fisher Scientific (1 mentions)
P h2ax, by Cell Signaling Technology (1 mentions)
Nupage gel, by Thermo Fisher Scientific (1 mentions)
P atm, by Cell Signaling Technology (1 mentions)
Dylight secondary antibodies, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Foxm1, by Cell Signaling Technology (1 mentions)
P atm, by Abcam (1 mentions)
Rad50, by Abcam (1 mentions)
Pnbs1, by Abcam (1 mentions)
Histone h3, by Wanlei (1 mentions)
γ h2ax, by Abcam (1 mentions)
Gapdh, by Abcam (1 mentions)
Bcl 2, by Abcam (1 mentions)
Bcl2 associated x (bax), by Abcam (1 mentions)
Nuclear and cytoplasmic protein extraction kit, by Beyotime (1 mentions)
Cleaved caspase 3, by Wanlei (1 mentions)
Caspase 3, by Wanlei (1 mentions)
Mre11, by Abcam (1 mentions)
2 protocols using p rad50
1
Western Blot Analysis of DNA Damage Signaling
2
Nucleoprotein Extraction and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Nucleoprotein was extracted using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology, China) according to the manufacturer’s instructions. Total protein was extracted using radioimmunoprecipitation buffer supplemented with the protease inhibitor PMSF. Western blotting was performed as described previously (27 (link)). Each blot was repeated in three separate experiments. Band intensities were measured using Fusion software and the data are presented as relative protein levels normalized to GAPDH or histone H3. The antibodies used in this study were as follows: FoxM1 and P-RAD50 (Cell Signaling Technology, Danvers, MA, USA); Bax, Bcl2, GAPDH, NBS1, P-NBS1, MRE11, P-MRE11, RAD50, ATM, P-ATM, and γH2AX (Abcam, Cambridge, UK); and caspase-3, cleaved caspase-3, and histone H3 (Wanleibio, China).
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