Chromatin Fractionation and Western Blot Analysis

Chromatin fractionation experiments were performed as previously described (23 (link)). Western blotting was performed using standard methods. Blots were incubated with primary antibodies against: rabbit anti-pCHK1(S345) (Cell Signalling Technology), mouse anti-CHK1 (Santa Cruz Biotechnology), rabbit anti-RAD51 (Bioss Antibodies), mouse anti-RPA32 (Calbiochem), rabbit anti-RPA70 (GeneTex), mouse anti-GAPDH (Millipore) and rabbit anti-Lamin B1 (Abcam). After incubations with horseradish peroxidase-linked secondary antibodies (1:20 000, Jackson Immunosciences), the blots were developed using the chemiluminescence detection kit WesternBright ECL HRP substrate (Advansta) according to the manufacturer's instructions. Quantification was performed on scanned images of blots using the Image Lab software, and the values shown on the graphs represent normalization of the protein content evaluated through LaminB1 or GAPDH immunoblotting.

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Aiello F.A., Palma A., Malacaria E., Zheng L., Campbell J.L., Shen B., Franchitto A, & Pichierri P. (2019). RAD51 and mitotic function of mus81 are essential for recovery from low-dose of camptothecin in the absence of the WRN exonuclease. Nucleic Acids Research, 47(13), 6796-6810.

Publication 2019

rabbit anti-pCHK1(S345) mouse anti-CHK1 rabbit anti-RPA70 mouse anti-GAPDH rabbit anti-Lamin B1 WesternBright ECL HRP substrate

Anti b1 Antibodies Chemiluminescence Chromatin Fractionation Gapdh Lamin Mouse Protein Rabbit

Corresponding Organization : Istituto Nazionale Biostrutture e Biosistemi

Other organizations : City of Hope, Cancer Genetics (United States), California Institute of Technology

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Variable analysis

independent variables

Chromatin fractionation experiments

dependent variables

Protein levels of pCHK1(S345), CHK1, RAD51, RPA32, RPA70, GAPDH, and Lamin B1

control variables

Standard Western blotting methods
Incubation with primary antibodies
Incubation with horseradish peroxidase-linked secondary antibodies
Chemiluminescence detection using WesternBright ECL HRP substrate

controls

Positive control: Not specified
Negative control: Not specified

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