Figure 3: Generation of KCNJ5 monoclonal antibodies.
Monoclonal antibodies against KCNJ5 were produced by injection of mice with a synthetic peptide corresponding to the N-terminal portion of KCNJ5 (acetyl-36-ATDRTRLLAEGKKP-49-C). See methods for details. The specificity of antibodies KCNJ5–68-15 and KCNJ5–33-11 was validated by Western blotting of cell lysates of HEK 293T cells transduced with a tetracycline-inducible lentivirus containing the human KCNJ5 sequence (Panel A, uninduced [lanes 1 and 3] and tetracycline-induced [lanes 2 and 4]). KCNJ5 immunohistochemistry of adrenal cortex adjacent to an APA using KCNJ5–68-15 and KCNJ5–33-5 (Panel B). KCNJ5–68-15 resulted in staining of most of the cortical tissue with evident staining of nuclei (Panel B, left). KCNJ5–33-5 produced intense staining of the zone glomerulosa with clear localization to the plasma membrane (Panel B, right). Panel B scale bar = 100 μm.