Project description:Estrogen Receptor alpha (ERα) is a key driver of most breast cancers, and it is the target of endocrine therapies used in the clinic to treat women with ERα positive (ER+) breast cancer. The two methods ChIP-seq (chromatin immunoprecipitation coupled with deep sequencing) and RIME (Rapid Immunoprecipitation of Endogenous Proteins) have greatly improved our understanding of ERα function during breast cancer progression and in response to anti-estrogens. A critical component of both ChIP-seq and RIME protocols is the antibody that is used to pull down the bait protein. To date, most of the ChIP-seq and RIME experiments for the study of ERα have been performed using the sc-543 antibody from Santa Cruz Biotechnology. However, this antibody has been discontinued, thereby severely impacting the study of ERα in normal physiology as well as diseases such as breast cancer and ovarian cancer. Here, we compare the sc-543 antibody with other commercially available antibodies, and we show that 06-935 (EMD Millipore) and ab3575 (Abcam) antibodies can successfully replace the sc-543 antibody for ChIP-seq and RIME experiments.
Project description:Keratinocyte carcinomas (BCC and cSCC) are the most common cancers worldwide. cSCC is considered as the most prevalent metastatic skin malignancy and its incidence is increasing globally. The complement system is a fundamental part of the host immune defense. It is composed of three distinct pathways (classical, alternative and lectin) that get activated sequentially. However, the process of complement activation is rigorously regulated by a series of soluble and membrane-bound inhibitor proteins that protect the host cells from lytic damage. One of these soluble negative regulators is complement factor I (CFI) which is an 88 kDa serine protease that hampers all three complement pathways through blockade of C3- and C5- convertases by cleaving C3b and C4b. Oligonucleotide array (Affymetrix)-based analysis of normal human epidermal keratinocytes (NHEKs; n=5), primary (Prim. cSCC; n=5) and metastatic (Met. cSCC; n=3) cSCC cell lines as well as next-generation-sequencing (SOLiD) based transcriptome profiling of NHEKs (n=4), primary (Prim. cSCC; n=5) and metastatic (Met. cSCC; n=3) cSCC cell lines revealed marked overexpression of CFI in cSCC cells compared to NHEKs (GSE66368 and GSE66412 respectively). Furthermore, we have previously shown that knockdown of CFI inhibits proliferation and migration of cSCC cells and potently impedes growth of cSCC xenograft tumors in vivo. In these respects, we intended to further investigate the functional role and molecular mechanism of CFI in cSCC progression via analyzing the mRNA expression profile of CFI in cSCC cells.