Detection of Human Phospho-53BP1 (S25) by Western Blot. Western blot shows lysates of K562 human chronic myelogenous leukemia cell line untreated |
(-) or exposed (+) to 50 J/m2 UV-C and allowed 1 hour recovery time. PVDF membrane was probed with 1 µg/mL Rabbit Anti-Human Phospho-53BP1 (S25) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3405) followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # HAF008). A specific band for Phospho-53BP1 (S25) was detected at approximately 450 kDa (as indicated). The phospho-specificity of this antibody was supported by decreased labeling following treatment with 600 U lambda -phosphatase ( lambda -PPase) for 1 hour. This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.
The p53-binding protein 1 (53BP1) localizes to sites of damaged DNA and is phosphorylated by the ATM checkpoint kinase. 53BP1 is required for phosphorylation of other downstream ATM substrates, such as p53 and SMC-1, and is itself a substrate for the ATM kinase. ATM phosphorylates 53BP1 at S25, and this phosphorylation aids in 53BP1 nucleating a DNA damage response protein complex.
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