Fig. 2.

Response of skeletal muscle transporters to K⁺-deficient state assayed by immunoblot of muscle homogenates. 1× and ½× amounts were assayed to confirm linearity of detection system (one amount is shown); assay and antibody details are provided in Table 1. A: individual values plotted as relative abundance normalized to mean of the 1% K (1K) group = 1; data are displayed as means ± SE; n = 6/group were assayed for all but NKAβ2 isoform (n = 3) because this noncommercial antibody degraded upon reuse and we did not identify a replacement for detecting mouse NKAβ2. Na⁺,K⁺-ATPase αβ subunits (NKAα1, α2, β1, β2); Na⁺,K⁺,2Cl⁻ cotransporter isoform 1 (NKCC1); NKCC1 phosphorylated at Thr212 and Thr 217 (NKCC1p); K⁺-Cl⁻ cotransporter isoform 3 (KCC3) glycosylated at 150 kDa and core at 100 kDa; KCC3 phosphorylated at Thr 1048 (KCC3p); Ste20/SPS-1-related proline-alanine-rich kinase (SPAK); SPAK phosphorylated at Ser 373 (SPAKp); oxidative stress-responsive kinase 1 phosphorylated at Ser 325 (OSR1p). B: typical immunoblots and normalized values (1K mean = 1), means ± SE calculated for n = 6/group (n = 3 for NKAβ2) and n = 3/group shown for simplicity. Molecular weights (MW; kDa), –br and –kid indicate mouse brain and kidney homogenate positive controls, respectively, and KO indicates a lane loaded with skeletal muscle homogenate from a SPAK-KO mouse (E. Delpire, Vanderbilt). Comparisons were performed using GraphPad Prism parametric unpaired Student’s t test and P values are provided.