ATR biochemical assays used human recombinant tagged proteins ATR/ATRIP (Eurofins, Item#14–953) purified from mammalian cells. Assays were conducted using glutathione S-transferase–tagged p53 substrate (Enzo Lifesciences; catalog no. BML-FW9370–0050) and 3 μmol/L ATP in 50 mmol/L HEPES, 0.01% Brij, 0.1 mmol/L EGTA, and 1% glycerol. Phosphorylated substrate was incubated with anti-phospho-p53 (Ser15) antibody (NEB; catalog no. 9284), donor (Perkin Elmer, 6765301), and acceptor (Perkin Elmer, 670137M) beads for 4 to 5 hours in 60 mmol/L EDTA, 50 mmol/L Tris pH 7.4 with 1.0 mg/mL BSA. Phosphorylation was quantified by fluorescence measured in a BMG Polarstar plate reader (excitation 680 nm, emission 520–620 nm). Standard PIKK and related kinase assays (Kinase Profiler) were performed at Eurofins Discovery, using standard assay protocols. See Supplementary Methods.
Atr atrip
Manufactured by Eurofins
The ATR/ATRIP is a laboratory equipment designed for attenuated total reflection (ATR) analysis. It provides a reliable and efficient method for infrared spectroscopy measurements. The core function of the ATR/ATRIP is to facilitate the acquisition of infrared spectra from solid, liquid, or semi-solid samples.
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2 protocols using atr atrip
1
ATR Kinase Activity Assay with p53
2
Purification of FA Protein Complexes
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Table 1 outlines the plasmids and bacmids used in this study and their derivation. Plasmids were propagated using NEB-10-beta competent cells and purified using Monarch miniprep kits (NEB). Bacmids were generated using the Multibac system (Berger et al., 2004 (link)) and purified using alkaline lysis method followed by isopropanol precipitation and resuspension in TE.
Human FANCI:FANCD2 complex and Avi-ubiquitin was purified as described in Tan et al. (2020) . Xenopus laevis (frog) FANCI:FANCD2, human FANCB:FANCL:FAAP100, FANCC:FANCE:FANCF and UBE2T were expressed and purified as described in van Twest et al. (2017) (link). USP1:UAF1, HA-ubiquitin and UBE1 were purchased from Boston Biochem. ATR-ATRIP was purchased from Eurofins DiscoverX. Lambda phosphatase was purchased from New England Biolabs.
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Human FANCI:FANCD2 complex and Avi-ubiquitin was purified as described in Tan et al. (2020) . Xenopus laevis (frog) FANCI:FANCD2, human FANCB:FANCL:FAAP100, FANCC:FANCE:FANCF and UBE2T were expressed and purified as described in van Twest et al. (2017) (link). USP1:UAF1, HA-ubiquitin and UBE1 were purchased from Boston Biochem. ATR-ATRIP was purchased from Eurofins DiscoverX. Lambda phosphatase was purchased from New England Biolabs.
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