Figure 5. Validation of anti-phospho-Ser¹²⁶ antibody.

Sequential immunoblotting with the phospho-Ser¹²⁶ (pSer126) antibody and the T4 antibody demonstrates specificity of the phospho-antibody for the NKCC2 isoform in its phosphorylated state. (A) GFP–NKCC2^N−term or GFP–NKCC2^{N−termS126A} were immunoprecipitated from transfected HEK-293 lysates. λ-Phosphatase treatment was used to de-phosphorylate the protein before in vitro phosphorylation with AMPK. (B) T4 immunoprecipitations from MMDD1 cell lysates were performed.