Immunoblotting of Fanconi Anemia Proteins

Immunoblots were performed as described (Bourseguin et al., 2016 (link)). The antibodies used were: mouse monoclonal anti-FANCM antibody (CV5.1, Novus biologicals, Abingdon, UK) (RRID:AB_2716711), mouse monoclonal anti-FANCD2, (Santa-Cruz Biotechnology, Dallas, Texas, USA, SC-20022) (RRID:AB_2278211), rabbit anti-FANCA (Abcam) (Bethyl Laboratories, Montgomeryn Texas, USA, Cat# A301-980A RRID:AB_1547945), mouse anti-vinculin (Abcam) (RRID:AB_11156698), Rabbit anti-CHK1 antibody from Santa-Cruz Biotechnology (SC-8408) (RRID:AB_627257), mouse monoclonal anti-Phospho-CHK1 (Ser317) antibody from Cell Signaling Technology (#2344) (RRID:AB_331488), monoclonal anti-phospho-H2AX (Ser139) antibody from Millipore (RRID:AB_309864). To transiently deplete FANCM, HEK293 cells (RRID:CVCL_0045) were transfected with 20 nmol/L of small INTERFERing RNA (siRNA) targeting FANCM, 5'-GGC-UAC-GUC-CAG-GAG-CGC-3' with 8 μL INTERFERin (Polyplus) in Opti-MEM. The protein bands were visualized and recorded using an ImageQuant apparatus. Western blot quantifications were performed using densitometry measures and the ImageJ software.

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Fouquet B., Pawlikowska P., Caburet S., Guigon C., Mäkinen M., Tanner L., Hietala M., Urbanska K., Bellutti L., Legois B., Bessieres B., Gougeon A., Benachi A., Livera G., Rosselli F., Veitia R.A, & Misrahi M. (2017). A homozygous FANCM mutation underlies a familial case of non-syndromic primary ovarian insufficiency. eLife, 6, e30490.

Publication 2017

rabbit anti-FANCA mouse anti-vinculin INTERFERin

A301 Anti antibody Antibodies Antibody Biologicals Densitometry Fanca Fancd2 Hek293 cells Immunoblots Monoclonal antibody Mouse Novus Protein Rabbit Sirna Vinculin Western blot

Corresponding Organization :

Other organizations : Université Paris-Sud, Université Paris-Saclay, Institut Gustave Roussy, Centre National de la Recherche Scientifique, Université Paris Cité, Institut Jacques Monod, Inserm, Délégation Paris 7, Turku University Hospital, Sorbonne Paris Cité, CEA Paris-Saclay, Commissariat à l'Énergie Atomique et aux Énergies Alternatives, Hôpital Necker-Enfants Malades, Centre de Recherche en Cancérologie de Lyon, Assistance Publique – Hôpitaux de Paris

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Variable analysis

independent variables

Depletion of FANCM using small interfering RNA (siRNA)

dependent variables

Protein levels of FANCM, FANCD2, FANCA, Chk1, phosphorylated Chk1, and phosphorylated H2AX

control variables

Vinculin (used as a loading control)

controls

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

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