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Rabbit Polyclonal Antibodies to Detect C7orf49 Proteins Toward Understanding Double Strand Break Repair
Author(s) -
Englund Bailey
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.906.5
Subject(s) - biology , microbiology and biotechnology , polyclonal antibodies , hek 293 cells , recombinant dna , dna , dna repair , antibody , genetics , gene
DNA double‐strand breaks are a highly cytotoxic form of DNA damage. Non‐homologous end joining (NHEJ) is an important mechanism that repairs double‐strand breaks in DNA. Accurate function of NHEJ protects genome integrity; however, malfunctions within the mechanism can lead to genomic changes resulting in carcinogenesis. The basic requirements for the NHEJ process have been determined, but there are still questions about the step‐wise assembly of the system. Our preliminary data indicates chromosome 7 open reading frame 49 (C7orf49) produces short open reading frame (sORF)‐encoded proteins that may participate in non‐homologous end joining (NHEJ). C7orf49 encodes three alternatively spliced protein isoforms, which will be referred to as spliced forms 1, 2 and 3 (SF‐1, SF‐2 and SF‐3, respectively). Transcriptome analysis and ribosomal profiling indicate that the C7orf49 locus is both transcribed and translated in human cells; but it is presently unclear: 1) whether endogenously expressed C7orf49‐encoded proteins are stable or degraded, 2) how abundant C7orf49‐encoded proteins are, 3) what isoforms are expressed, and 4) how DNA damage affects the abundance of C7orf49‐encoded proteins. The goal of this project is to determine whether our anti‐C7orf49 antibodies, rabbit polyclonal antibodies, can be used to detect C7orf49‐encoded proteins in human cell lysates. Antibodies were tested via Western blot under variable conditions with both wild type and C7orf49‐deficient HEK293 cells. C7orf49 antisera recognized SF‐1 and SF‐2 as recombinant purified proteins or when ectopically expressed in HEK293 cells. Compared to pre‐immune sera, immune sera recognized bands in wild type cell extracts, which may be encoded by C7orf49. Future experiments will investigate which isoforms are expressed, and what affects DSB‐inducing drugs have on C7orf49‐encoded protein abundance, to help determine how these proteins contribute to NHEJ. Support or Funding Information Support received from Leslyn Hanakahi and Matthew Summerlin at University of Illinois Chicago ‐ Rockford