Phospho chk1 ser317

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The Phospho-Chk1 Ser317 is a laboratory reagent used to detect and measure the phosphorylation of the Chk1 protein at the serine 317 residue. This phosphorylation event is an important cellular response to DNA damage and replication stress.

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2 protocols using phospho chk1 ser317

Cell Lysis and Protein Quantification

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Cells were harvested and subsequently lysed for 30 min in RIPA (25 mM Tris-HCl pH 7.6; 150 mM NaCl; 1% NP-40; 1% Sodiumdeoxycholate and 0.1% SDS) or ELB (150 mM NaCl; 50 mM Hepes pH7.5; 5 mM EDTA; 0.1% NP-40) containing protease inhibitors (Complete, Roche). Protein concentrations were measured using the BCA protein assay kit (Pierce).
The primary antibodies used were rabbit polyclonal phospho-Chk1 Ser317 (Bethyl), mouse monoclonal Chk1 (G4; Santa Cruz), goat polyclonal CDK4 (C22; Santa Cruz), rabbit polyclonal p21 (C19; Santa Cruz), mouse monoclonal p27 (BD Transduction Laboratory), goat polyclonal Rb (C15; Santa Cruz); rabbit polyclonal Rbl1 (C18; Santa Cruz), mouse monoclonal Rbl2 (CAS14; Lab Vision), mouse monoclonal p53 (IMX25; monosan; for detection of mouse p53), mouse monoclonal p53 (DO-1; BD Biosciences; for detection of human p53), γ-tubulin (GTU-88; Sigma), rabbit polyclonal Cyclin D1 (Santa Cruz; H296) and goat polyclonal CDK4 (C22; Santa Cruz). Secondary antibodies used were IR Dye 800CW Goat anti-Mouse IgG, Goat anti-Rabbit IgG and Donkey anti-Goat IgG (Licor) and HRP-conjugated Goat anti-Mouse and Goat anti-Rabbit (Dako).

Benedict B., van Harn T., Dekker M., Hermsen S., Kucukosmanoglu A., Pieters W., Delzenne-Goette E., Dorsman J.C., Petermann E., Foijer F, & te Riele H. (2018). Loss of p53 suppresses replication-stress-induced DNA breakage in G1/S checkpoint deficient cells. eLife, 7, e37868.

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DNA Damage Response Signaling Immunoblotting

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Immunoblot analysis was performed as described in detail previously [21 (link),22 (link)]. Primary antibodies against ATM (Bethyl Laboratories, Montgomery, TX, USA), phospho-ATM Ser1981 (Cell Signaling Technologies, Danvers, NJ, USA), ATR (Bethyl Laboratories), phospho-ATR Ser428 (Cell Signaling Technologies), p53 (Cell Signaling Technologies), phospho-p53 Ser15 (Cell Signaling Technologies), SMC1 (Bethyl Laboratories), phospho-SMC1 Ser966 (Bethyl Laboratories), Chk1 (G-4; Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-Chk1 Ser317 (Bethyl Laboratories), phospho-Chk1 Ser345 (Cell Signaling Technologies), Chk2 (H-300; Santa Cruz), phospho-Chk2 Thr68 (Cell Signaling Technologies), Wip1 (PPM1D; Bethyl Laboratories), Cdc25A (F-6; Santa Cruz) and GAPDH (Cell Signaling Technologies) were used for immunoblots.

Tatewaki N., Konishi T., Nakajima Y., Nishida M., Saito M., Eitsuka T., Sakamaki T., Ikekawa N, & Nishida H. (2016). Squalene Inhibits ATM-Dependent Signaling in γIR-Induced DNA Damage Response through Induction of Wip1 Phosphatase. PLoS ONE, 11(1), e0147570.

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