Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Diverse BRCA1 and BRCA2 Reversion Mutations in Circulating Cell-Free DNA of Therapy-Resistant Breast or Ovarian Cancer

doi: 10.1158/1078-0432.CCR-17-0544

Figure Lengend Snippet: A, CT images during the course of therapy of breast cancer patient L031 demonstrating the initial response and subsequent progression of the lesions. Plasma samples were obtained before and after treatment with the PARP inhibitor Talazoparib and after Capecitabine therapy (top). Mutant allele frequencies of two somatic BRCA2 reversion mutations identified by targeted massively parallel sequencing were assessed in two independent analyses in the plasma samples pre- and post PARP inhibitor treatment using targeted amplicon sequencing. B, 293T cells transfected with HA-BRCA2 wild-type (WT), HA-BRCA2 c.407delA germline mutant (GM) and HA-BRCA2 c.402_413delTCTAAATTCTTG somatic reversion-mutant plasmids Rev). Western blot performed using an anti-HA antibody revealed that the HA-BRCA2Rev was translated into mutant protein (predicted 3414AA) with a molecular weight similar to that of the wild-type protein (3418AA). The HA-BRCA2GM protein length is predicted to be 150AA. Immunoprecipitation of HA-BRCA2Rev and wild-type HA-BRCA2 revealed that HA-BRCA2Rev protein displays proficient interactions with PALB2 and RAD51 similar to that of the wild-type BRCA2 protein. AA, amino acid.

Article Snippet: After three washes with wash buffer (0.5% NP40, 25 mM Tris pH 7.5, 150 mM NaCl, 0.5 mM EDTA), the beads were boiled in SDS sampling buffer, followed by western blotting with antibodies against HA (Santa Cruz), PALB2 (Novus Biologicals) and RAD51 (Santa Cruz).

Techniques: Clinical Proteomics, Mutagenesis, Sequencing, Amplification, Transfection, Western Blot, Molecular Weight, Immunoprecipitation