Immunoblotting Analysis of DNA Damage Signaling

BMDM and MEFs were lysed in RIPA buffer and whole cell lysates were generated with LDS sample buffer (Invitrogen) supplemented with dithiothreitol (DTT). For analysis of culture supernatants, protein was precipitated with 7.2% w/v trichloroacetic acid (TCA) (Sigma) followed by two acetone washes. Immunoblotting was carried out as previously described (Helmink et al., 2011 (link)). Primary antibodies used were anti-γ-H2AX clone JBW301 (Millipore) (RRID:AB_309864), anti-H2AX (Millipore) (RRID:AB_2233033), anti-phospho-KAP-1 (Bethyl Laboratories) (RRID:AB_669740), anti-KAP-1 (GeneTex) (RRID:AB_372041), anti-caspase 1 (p20, Casper-1) (Adipogen) (RRID:AB_2490248), anti-DNA-PKcs (Invitrogen), anti-Ku70 (Cell Signaling Technology), anti-Ku80 (Cell Signaling Technology) (RRID:AB_2257526), anti-ATM clone MAT3 (Sigma), anti-Mre11 (Novus) (RRID:AB_10077796), anti-Nbs1 (Abcam) (RRID:AB_777006), anti-Rad50 (Abcam) (RRID:AB_2176935), anti-ATR (Novus) (RRID:AB_10003234), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Sigma). Secondary reagents were horseradish peroxidase–conjugated anti–mouse IgG (Promega) or horseradish peroxidase–conjugated anti- rabbit IgG (Cell Signaling Technology).

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Morales A.J., Carrero J.A., Hung P.J., Tubbs A.T., Andrews J.M., Edelson B.T., Calderon B., Innes C.L., Paules R.S., Payton J.E, & Sleckman B.P. (2017). A type I IFN-dependent DNA damage response regulates the genetic program and inflammasome activation in macrophages. eLife, 6, e24655.

Publication 2017

LDS sample buffer anti-γ-H2AX clone JBW301 anti-H2AX anti-phospho-KAP-1 anti-DNA-PKcs anti-Ku70 anti-Ku80 anti-Mre11 anti-Rad50 anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) horseradish peroxidase–conjugated anti–mouse IgG horseradish peroxidase–conjugated anti- rabbit IgG

Acetone Anti dna Anti igg Antibodies Buffer Caspase 1 Cell Clone Dithiothreitol Dna pkcs Glyceraldehyde 3 phosphate dehydrogenase Horseradish peroxidase Kap 1 Ku70 Mouse Novus Pkcs Promega Protein Rabbit Rad50 Ripa Trichloroacetic acid

Corresponding Organization :

Other organizations : Cornell University, Washington University in St. Louis, National Institute of Environmental Health Sciences

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Variable analysis

independent variables

Cell type (BMDM and MEFs)

dependent variables

Levels of proteins (γ-H2AX, H2AX, phospho-KAP-1, KAP-1, caspase 1, DNA-PKcs, Ku70, Ku80, ATM, Mre11, Nbs1, Rad50, ATR) in whole cell lysates and culture supernatants

control variables

RIPA buffer for cell lysis
LDS sample buffer (Invitrogen) supplemented with dithiothreitol (DTT) for preparing whole cell lysates
7.2% w/v trichloroacetic acid (TCA) (Sigma) for protein precipitation from culture supernatants, followed by two acetone washes
Immunoblotting procedure as previously described (Helmink et al., 2011)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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