Invention novelty: JHU researchers have generated PS120/FLAG-NF2 cells with human WTNHE3 expression (no TAG), stable human R799C mutant NHE3 expression (no tag), and with stable rabbit WT NHE3 expression (no tag).
Value Proposition: NHE null fibroblasts expression in different species and polymorphisms of NHE3 to be used for functional studies. R799C is a mutant that occurs in a significant percent of certain world populations and has been identified in the NHE3 gene. JHU researchers have generated PS120/FLAG-NF2 cells with NHE3 expression.
Technical Details:Cell Lines, Cell Culture, and Transfection: Chinese hamster lung fibroblasts PS120 cells were used to generate HA-NHE3 stably expressing cells by transient transfection of pcDNA3.1-HA-NHE3 and G418 selection. Stable PS120 cell lines expressing FLAG-NHERF proteins' generated similarly. Opossum kidney proximal tubular OK cells were cultured in plastic dishes and transfected at 90°; confluency for co-immunoprecipitation or seeded on glass-slides and transfected on the second day post confluency immunostaining. Transient transfection was performed with 0.4 µg of each plasmid using 0.8 µI of Lipofectamine 2001 (Invitrogen) per cm2 of growth surface. Then cells were grown in complete medium and used for experiments 48 h later. The human colon cancer Caco-2/bbe cell line was grown on collagen-coated Transwell filter membranes (EMD Millip Darmstadt, Germany) as previously described (23). Caco-2-NHERF3-KD cells were established previously by infectic Caco-2/bbe cells with lentivirus-shRNA-puromycin (24).
Co-IP: Cell lysates were prepared with the lysis buffer (25 mM HEPES, pH 7.4, 150 mM NaCI, 50 mM NaF, 1 mM Na3VO4, 0.5 Triton X-100, and protease inhibitors). Anti-FLAG M2 magnetic beads were washed with this buffer three times. 0.5 mg of each cell lysate were mixed with 5 µI of anti-FLAG M2 magnetic beads and incubated at 4ºC for 3 h on a rotating shaker. Beads were washed with the same lysis buffer 4 times and eluted with 1.5x Laemmli sample buffer without ß-mercaptoethanol. The input and elution samples were analyzed by SDS-PAGE and Western blotting.
Publication(s)/Associated Cases: J. Biol. Chem. 2014 289: 20039-20053.
