Technical Reference #1880

1880.

Epidermal growth factor activates Na+/H+ exchanger in podocytes through a mechanism that involves Janus kinase and calmodulin Sonya D. Coaxum; Maria N. Garnovskaya; Monika Gooz; Aleksander Baldys; John R. Raymond, Medical and Research Services of the Ralph H. Johnson VA Medical Center, Biochimica et Biophysica Acta, 1793(1880), (2009)

Keywords:
Cytosensor microphysiometer; ECAR; Phosphorylation; AG1478

Materials & Methods:
Immunolabeling was performed as previously described [34]. Cells were seeded in 35-mm collagen-coated glass-bottom culture dishes (MatTek Corporation) and fixed with 2% paraformaldehyde 4% sucrose in phosphate-buffered saline (PBS) for 10 min at room temperature. Subsequently cells were permeabilized with 0.3% Triton X-100 (Sigma) in PBS for 5 min following which nonspecific binding sites were blocked with 2% fetal calf serum 2% BSA and 0.2% gelatin in PBS for 1 h. Incubations with the appropriate dilutions of primary and secondary antibodies (as directed by the manufacturer) were performed in blocking solution. The primary and secondary antibodies used were: anti-WT1 (1:50) (Santa Cruz Biotechnology); antisynaptopodin (Research Diagnostics Inc.) and Alexa Fluor 488 goat anti-mouse IgG (Molecular Probes). Confocal microscopy was performed using a Zeiss LSM 510 META laser-scanning microscope (Carl Zeiss Inc.).

Microscopic Technique
Confocal Microscopy

Cell Type(s)
podocyte cell line