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Antibodies Against Sodium‐Dependent Cation Chloride Cotransporters in the Yellow Fever Mosquito Aedes aegypti
Author(s) -
Viel Kenyatta CMF,
DeBrosse Adrienne C.,
Riley Grace F.,
Piermarini Peter M.,
Gillen Christopher M.
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.719.6
Subject(s) - cotransporter , biology , antiserum , malpighian tubule system , polyclonal antibodies , antibody , microbiology and biotechnology , blot , peptide sequence , secretion , aedes aegypti , biochemistry , sodium , midgut , gene , chemistry , larva , genetics , botany , organic chemistry
Sodium‐dependent cation‐chloride cotransporters (CCCs) participate in ion secretion and absorption across animal phyla. The genome of the yellow fever mosquito Aedes aegypti has three genes with sequence similarity to vertebrate sodium‐dependent CCCs. One of these sequences (AAEL006180) codes for a protein with 50% identity to the human secretory Na‐K‐Cl cotransporter NKCC1 and 70% identity to a Drosophila melanogaster Na‐K‐Cl cotransporter with a role in secretion by Malpighian tubules. The other two of these sequences (AAEL009888 and AAEL009886) code for proteins with approximately 40% identity to the human Na‐K‐Cl cotransporter NKCC1 and approximately 55% identity to uncharacterized proteins in Drosophila and other insects. To begin evaluating the role of the proteins coded for by AAEL009888 and AAEL009886, we conducted experiments to determine their developmental and tissue expression patterns. Polyclonal antibodies were developed by injecting two rabbits with two peptide sequences from each paralog. Antiserum from both rabbits reacted with proteins in larvae, adult male, and adult female whole body tissues, as well as in adult Malpighian tubules and larval anal papillae, with the most prominent bands above 150 kDa size markers on 7.5% SDS‐PAGE gels. To develop paralog‐specific antibodies, we affinity purified the antiserum against peptides from either the AAEL009888 or AAEL009886 sequences. We confirmed specificity of these antibodies with peptide competition experiments on dot blots. Similar to the unpurified serum, the affinity purified antibodies reacted with proteins above 150 kDa on Western blots of lysates and membrane preps from whole body samples of larvae and adults. We are testing the effectiveness of the affinity purified antibodies on Western blots of isolated tissue samples and on formalin‐fixed paraffin‐embedded sections of larvae. Support or Funding Information NSF‐IOS‐1557230