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Immunoblotting and Co-immunoprecipitation of DNA Damage Proteins

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Whole-cell extracts were prepared and subjected to immunoblotting as previously described (23 (link)). Immunoblotting was performed using antibodies against pS1981 ATM (AF1655) from R&D Systems; ATM (A300-299A), pS824-KAP1 (A300-767A), KAP1 (A300-274A), pS966-SMC1 (A300-050A), SMC1 (A300-055A), pS4/S8-RPA2 (A300-245A), and RECQL1 (A300-450A) from Bethyl Laboratories; pS345-CHK1 (2341) from Cell Signaling Technology; CHK1 (sc-8408) and PARP1 (sc-8007) from Santa Cruz Biotechnology; H2A (07-146), γ-H2AX (05-636), RPA2 (NA18), and HA (H9658) from Merck; pS343-NBS1 (47272) from Abcam; and Nbs1 (GTX70224) from GeneTex. Co-immunoprecipitation was carried out as previously described (23 (link)), with the exception that EDTA was excluded, and 250 units/mL benzonase (Merck) and 1.5 mM MgCl₂ were added to the lysis buffer. Anti-RECQL1 antibody (5 μg, A300-450A) or a nonspecific IgG (Dako) coupled with protein A agarose beads (GE Healthcare) were used to isolate protein complexes from 5 mg whole-cell extract.

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Abu-Libdeh B., Jhujh S.S., Dhar S., Sommers J.A., Datta A., Longo G.M., Grange L.J., Reynolds J.J., Cooke S.L., McNee G.S., Hollingworth R., Woodward B.L., Ganesh A.N., Smerdon S.J., Nicolae C.M., Durlacher-Betzer K., Molho-Pessach V., Abu-Libdeh A., Meiner V., Moldovan G.L., Roukos V., Harel T., Brosh RM J.r, & Stewart G.S. (2022). RECON syndrome is a genome instability disorder caused by mutations in the DNA helicase RECQL1. The Journal of Clinical Investigation, 132(5), e147301.

Publication 2022

RECQL1 (A300-450A) benzonase protein A agarose beads

Agarose Anti antibody Antibodies Benzonase Buffer Cell extract Co immunoprecipitation Edta Kap1 Mgcl2 Parp1 Protein Protein a Rpa2

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Variable analysis

independent variables

Antibodies used for immunoblotting

dependent variables

Protein expression and phosphorylation levels

control variables

Whole-cell extracts were prepared and subjected to immunoblotting as previously described (23)
Co-immunoprecipitation was carried out as previously described (23), with the exception that EDTA was excluded, and 250 units/mL benzonase (Merck) and 1.5 mM MgCl2 were added to the lysis buffer
Anti-RECQL1 antibody (5 μg, A300-450A) or a nonspecific IgG (Dako) coupled with protein A agarose beads (GE Healthcare) were used to isolate protein complexes from 5 mg whole-cell extract

negative controls

Nonspecific IgG (Dako) coupled with protein A agarose beads (GE Healthcare) were used to isolate protein complexes from 5 mg whole-cell extract

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