Transfection and Analysis of ACVR1 Mutants

ACVR1 mutations R206H and G328E were cloned into pcDNA3.1 by site-directed mutagenesis as previously described^{16 (link)} and transfected into primary cells QCTBR059 and SU-DIPG-VI using lipofectamine (Invitrogen), with protein collected after 24 hours using standard procedures. Western blots were carried out for anti-FLAG HRP (#A8592, Sigma; 1:1000 dilution) and phosphorylated Smad1/5/8 (#9511, Cell Signalling; 1:1000) under standard conditions. Relative levels of phosphorylated Smad1/5/8 were measured by Image J software (National Institute of Mental Health, Bethesda, MD, USA).

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Taylor K.R., Mackay A., Truffaux N., Butterfield Y., Morozova O., Philippe C., Castel D., Grasso C.S., Vinci M., Carvalho D., Carcaboso A.M., de Torres C., Cruz O., Mora J., Entz-Werle N., Ingram W.J., Monje M., Hargrave D., Bullock A.N., Puget S., Yip S., Jones C, & Grill J. (2014). Recurrent activating ACVR1 mutations in diffuse intrinsic pontine glioma. Nature genetics, 46(5), 457-461.

Publication 2014

lipofectamine anti-FLAG HRP phosphorylated Smad1/5/8

Cells Dilution Dipg Lipofectamine Mutations Protein Site directed mutagenesis Western blots

Corresponding Organization :

Other organizations : Institute of Cancer Research, Institut Gustave Roussy, BC Cancer Agency, Howard Hughes Medical Institute, University of California, Los Angeles, Oregon Health & Science University, Hospital Sant Joan de Déu Barcelona, Hôpital d'Hautepierre, Children's Medical Research Institute, Stanford University, Great Ormond Street Hospital, University College London, Genomics England, University of Oxford, Hôpital Necker-Enfants Malades

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Variable analysis

independent variables

ACVR1 mutations R206H and G328E

dependent variables

Levels of phosphorylated Smad1/5/8

control variables

Protein collected after 24 hours using standard procedures
Western blots carried out for anti-FLAG HRP and phosphorylated Smad1/5/8 under standard conditions

controls

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

Annotations

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