DRA (human) : Turner Lab

Downregulated in adenoma (DRA) is a transmembrane glycoprotein that transports chloride ions across the cell membrane in exchange for bicarbonate ions. Expression is primarily within intestinal epithelia of ileum and colon, where DRA localizes to the apical membrane of colonocytes and some goblet cells. DRA is critical to chloride absorption, and mutations in the gene (SLC26A3) have been associated with congenital chloride diarrhea.

Antibody charactierzation and validation:
This antibody was produced by immunizing rabbits with the C-terminal 19 amino acids of human SLC26A3/DRA (accession number BC025671, sequence INTNGGLRNRVYEPVETKF) as desribed (Kumar et al., 2017*). Antibodies were affinity purified using the peptide antigen. The antibody recognizes human and mouse DRA as a single band on western blot and in frozen sections (Kumar et al., 2017). In formalin-fixed, parrafin-embedded tissues, the antisera recognized human, but not mouse, DRA. As further validation in formalin-fixed, paraffin-embedded tissues, localization on the apical surface of villus epithelium within the ileum was confirmed (right, upper image). The absence of detectable signal in human jejunum (right, lower image), where DRA is known to be absent, provides further evidence of antibody specificity in this application. DRA was also detected in human colon and, at very low levels, in duodenum, consistent with RNA expression data.

Immunostaining procedure conditions:
Antigen retrieval with Target Retrieval Solution, pH9 (Agilent Technologies, Cat. #S2367) for 40 minutes at 95°C.
Primary and secondary incubations performed using the following reagents:

Antibody	Source	Catalog #	Lot #	RRID#	Working Dilution/Concentration
Anti-DRA antibody*	Dudeja Lab				1:500
Anti-E Cadherin antibody [M168]	Abcam	ab76055	GR216035	AB_1310159	2.5 µg/ml
Anti-actin [MAC 237]	Turner lab	ab50591	GR168034	AB_867488	8 µg/ml


Alexa Fluor® 488 AffiniPure Donkey Anti-Mouse IgG (H+L)	Jackson Immunoresearch	715-545-151	118237	AB_2341099	1.5 µg/ml
Alexa Fluor® 594 AffiniPure F(ab’)₂ Fragment Donkey Anti-Rabbit IgG (H+L)	Jackson Immunoresearch	711-586-152	124065	AB_2340622	1.5 µg/ml
Alexa Fluor® 647 AffiniPure F(ab’)₂ Fragment Donkey Anti-Rat IgG (H+L)	Jackson Immunoresearch	712-606-153	123491	AB_2340696	1.5 µg/ml
Hoechst 33342	Life Technologies	H3570			10 µg/ml

*Generously provided by Pradeep K. Dudeja, Ph.D, University of Illinois College of Medicine.
Kumar A, Chatterjee I, Gujral T, Alakkam A, Coffing H, Anbazhagan AN, Borthakur A, Saksena S, Gill RK, Alrefai WA, Dudeja PK. Activation of Nuclear Factor-kappaB by Tumor Necrosis Factor in Intestinal Epithelial Cells and Mouse Intestinal Epithelia Reduces Expression of the Chloride Transporter SLC26A3. Gastroenterology 2017;153:1338-1350.