Pig Renal Na/K ATPase and Bcl‐2 Proteins: A Co‐Immuno‐Precipitation Study

Abstract

The Na/K ATPase (NKA) has at least two Bcl‐2 like sequences (Cell Physiol. Biochem. Cell Physiol Biochem 31: 257–276, 2013) and, in human lens epithelial cells, co‐immuno‐precipitates with the Bcl‐2 proteins BclXL and Bak (Am J. Physiol. Cell Physiol 2015: 308: C51–C60). Here we searched for immunochemical evidence of the presence of BH3 motif‐containing Bak co‐immuno‐precipitated with the well‐characterized pig renal NKA (PRNKA) (Klodos et al., Kidney Int. 62:2097–2100, 2002).ResultsPRNKA was prepared by the Klodos procedure omitting the final SDS purification step to preserve NKA activity at 0.45 μmol in 5.54 mg/ml. Mouse (ms) anti‐NKA (α6F) antibody (ab) showed the α1 subunit of ~110 kDa in immunoblots (IB) of the PRNKA input, in the protein‐G coated metal bead (PGCMB) ‐ α6F immuno‐pulldown, as well as in PGCMB‐ms and rabbit (rb) IgG pulldowns. A rb polyclonal anti‐human Bak BH3 motif ab (Cell Signaling #3814) together with a secondary (2nd) goat anti‐Rb Fc’ heavy (H) chain ab detected a low level staining of a ~27 kDa peptide in the PRNKA input and a prominent band in the α6F immuno‐pull down suggesting the presence of Bak in the enzyme preparation. IBs with the 2nd anti‐Fc’ H‐chain ab alone, however, not only detected IgG H chains in α6F ab‐, and ms‐ and rb‐IgG immuno‐pull downs, but unexpectedly also in PRNKA input. These findings suggest the presence of 1) Bak, and 2) NKA in PRNKA inputs and in immune and “non‐immune” pulldowns, with an absence of both proteins in ms and rb IgG input only; and 3) an apparent IgG contamination in PRNKA. Because of the latter, our efforts are now directed to discriminate further between Bak and L chains both of very close molecular mass. Whether the triple occurrence of NKA, Bak and IgG in all pull downs is IgG‐molecular specific but not species‐specific or coincidental due to non‐specific adsorption to the protein G beads, or both, is currently under study.Support or Funding InformationSupported by Wright State University Foundation to the Cell Biophysics Laboratory, by the Pharmacology and Toxicology Program to C.K.M., and by NIH to JH.