Rad53 Phosphorylation Measurement in DNA Damage

Rad53 phosphorylation was assessed as described previously [9 (link)]. Briefly, exponentially growing cells (2-4x10⁷ cells/ml) in mec1Δ sml1Δ or mec1Δ sml1Δ sae2Δ background were cultured in presence of 0.15% MMS for 90 min. MMS was inactivated upon addition of 5% sodium thiosulfate (final concentration) to the cultures. TCA-extracts were prepared and 10–20 μg protein extracts were run on a 7.5% SDS-PAGE, transferred to nitrocellulose membrane and FLAG-Rad53 was detected by western blot with FLAG M2 mAb (Sigma).

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Hohl M., Mojumdar A., Hailemariam S., Kuryavyi V., Ghisays F., Sorenson K., Chang M., Taylor B.S., Patel D.J., Burgers P.M., Cobb J.A, & Petrini J.H. (2020). Modeling cancer genomic data in yeast reveals selection against ATM function during tumorigenesis. PLoS Genetics, 16(3), e1008422.

Publication 2020

FLAG M2 mAb

Cells Membrane Nitrocellulose Phosphorylation Protein Sds page Sodium thiosulfate Western blot

Corresponding Organization : Washington University in St. Louis

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Variable analysis

independent variables

Presence or absence of MMS (0.15%)

dependent variables

Rad53 phosphorylation
FLAG-Rad53 detection by western blot

control variables

Cell culture conditions (exponentially growing cells, 2-4x10^7 cells/ml)
Genetic background (mec1Δ sml1Δ or mec1Δ sml1Δ sae2Δ)
Protein extract amount (10–20 μg)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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