title: Using a new Lrig1 reporter mouse to assess differences between two Lrig1 antibodies in the intestine.
abstract: Lrig1 is an intestinal stem cell marker important for epithelial homeostasis. However, the position of the Lrig1(+) population in the intestinal crypt has been debated, largely due to discrepant staining patterns using two Lrig1 antibodies. Here, we set out to decipher the differences between these Lrig1 antibodies to clarify their use for Lrig1-related studies. We confirmed that the commercially available Lrig1-R&D antibody stained the bottom third of the colonic crypt, whereas an independently generated Lrig1-VU antibody recognized a subset of anti-Lrig1-R&D(+) cells. Biochemically, we found that anti-Lrig1-VU recognized a non-glycosylated form of Lrig1; in contrast, anti-Lrig1-R&D recognized both glycosylated and non-glycosylated forms of Lrig1. In addition, we generated a reporter mouse (Lrig1-Apple) as an independent readout of Lrig1 transcriptional activity. Flow cytometry of isolated colonic epithelial cells from Lrig1-Apple mice demonstrated anti-Lrig1-R&D recognized mostly RFP-hi cells, while anti-Lrig1-VU recognized cells that were largely RFP-mid. Of note, by qRT-PCR, Lgr5 was expressed in the RFP-hi population, but not in the RFP-mid population. We conclude that anti-Lrig1-R&D appears to recognize all Lrig1(+) cells, while anti-Lrig1-VU recognizes a subpopulation of Lrig1(+) cells.
authors.affiliation: Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
authors.forename: Emily J
authors.initials: EJ
authors.lastname: Poulin
authors.affiliation: Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
authors.forename: Anne E
authors.initials: AE
authors.lastname: Powell
authors.affiliation: Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
authors.forename: Yang
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
authors.forename: Yina
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Franklin
authors.affiliation: Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA; Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA; Department of Veterans Affairs Medical Center, Nashville, TN 37232, USA. Electronic address: robert.coffey@vanderbilt.edu.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Coffey
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology*; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007422:Intestines / Q000166:cytology*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*
chemical_list: D000906:Antibodies; C101671:Lrig1 protein, mouse; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins