Correlation of GREB1 mRNA with protein expression in breast cancer: validation of a novel GREB1 monoclonal antibody.
Studies of gene regulated by estrogen in breast cancer 1 (GREB1) have focused on mRNA levels with limited evidence about GREB1 protein expression in normal and breast cancer cells. A monoclonal antibody that recognizes GREB1 protein in breast tissues could be applied to correlate protein expression with established mRNA expression data. A hybridoma expressing a murine monoclonal antibody targeting a 119 amino acid peptide specific to human GREB1 was generated. The novel monoclonal GREB1 antibody (GREB1ab) was validated for use in Western blotting as well as immunohistochemical (IHC) applications. GREB1ab detects a 216 kDa protein corresponding to GREB1 in estrogen receptor alpha (ERalpha+) breast cancer cells as well as ERalpha- breast cancer cells transduced with a GREB1 expression vector. GREB1ab specificity was verified using an ERalpha antagonist to prevent GREB1 induction as well as a silencing siRNA targeting GREB1 mRNA. GREB1ab was further validated for detection of GREB1 by IHC in breast cancer cell lines and breast tissue microarrays (TMA). ERalpha+ cell lines were observed to express GREB1 while ERalpha- cell lines did not express detectable levels of the protein. Using breast cancer tissue whole sections, IHC with the GREB1ab identified protein expression in ERalpha+ breast cancer tissue as well as normal breast tissue, with little GREB1 expression in ERalpha- breast cancer tissue. Furthermore, these data indicate that GREB1 mRNA expression correlates well with protein expression. The novel monoclonal GREB1ab is specific for GREB1 protein. This antibody will serve as a tool for investigations focused on the expression, distribution, and function of GREB1 in normal breast and breast cancer tissues.
Department of Medicine, Miller School of Medicine, University of Miami, Miami, FL, USA. jhnatyszyn@med.miami.edu
H J
HJ
Hnatyszyn
M
M
Liu
A
A
Hilger
L
L
Herbert
C R
CR
Gomez-Fernandez
M
M
Jorda
D
D
Thomas
J M
JM
Rae
D
D
El-Ashry
M E
ME
Lippman
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics; D000918:Antibody Specificity; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D045744:Cell Line, Tumor; D004958:Estradiol / Q000378:metabolism; D004965:Estrogen Antagonists / Q000494:pharmacology; D047628:Estrogen Receptor alpha / Q000037:antagonists & inhibitors / Q000378:metabolism; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D006825:Hybridomas; D007150:Immunohistochemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D009363:Neoplasm Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D015203:Reproducibility of Results; D046888:Tissue Array Analysis
D000911:Antibodies, Monoclonal; D004958:Estradiol; D004965:Estrogen Antagonists; D047628:Estrogen Receptor alpha; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D018719:Erb-b2 Receptor Tyrosine Kinases; C416830:GREB1 protein, human; C508053:ERBB2 protein, human