I am trying to look at phospho ser 23 NKA alpha 1 in rat kidney tissue. I have used antibodies from cell signaling but my band appears around 120 kDa instead of the predicted 100 kDa on the data sheet. Has anyone else run this antibody and seen that it runs a little higher?
Please take a look at the following article
J Physiol 586.22 (2008) pp 5337–5348
Na+,K+-ATPase is modulated by angiotensin II in diabetic rat kidney – another reason for diabetic nephropathy?
Andrea Fekete
1, Klara Rosta2, LaszloWagner3, Agnes Prokai1, PeterDegrell3, Eva Ruzicska4, Edit Vegh2, Miklos Toth5, Katalin Ronai2, Krisztina Rusai1, Aniko Somogyi4, Tivadar Tulassay1,6, Attila J. Szabo1and Agota Ver2
Angiotensin II (ANGII) plays a central role in the enhanced sodium reabsorption in early type 1 diabetes inman and in streptozotocin-induced (STZ) diabetic rats. This study investigates the effect of untreated STZ-diabetes leading to diabetic nephropathy in combination with ANGII treatment, on the abundance and localization of the renal Na+,K+-ATPase (NKA), a major contributor of renal sodium handling. After 7 weeks of STZ-diabetes (i.v. 65 mg kg−1) a subgroup of control (C) and diabetic (D7)Wistar rats were treated with ANGII (s.c. minipump 33μg kg−1 h−1 for 24 h; CA and D7A). We measured renal function and mRNA expression, protein level, Serin23 phosphorylation, subcellular distribution, and enzyme activity of NKA α-1 subunit in the kidney cortex. Diabetes increased serum creatinine and urea nitrogen levels (C versus D7), as did ANGII (C versus CA, D7 versus D7A). Both diabetes (C versus D7) and ANGII increased NKA α-1 protein level and enzyme activity (C versus CA, D7 versus D7A). Furthermore, the combination led to an additive increase (D7 versus D7A,CAversus D7A).NKA α-1 Ser23 phosphorylation was higher both inD7and ANGII-treated rats in the non-cytoskeletal fraction, while no signal was detected in the cytoskeletal fraction. Control kidneys showed NKA α-1immunopositivity on the basolateral membrane of proximal tubular cells,while othD7and ANGII broadened NKA immunopositivity towards the cytoplasm. Our study demonstrates that diabetes mellitus (DM) increases the mRNA expression, protein level, Ser23 phosphorylation and enzyme activity of renal NKA, which is further elevated by ANGII. Despite an increase in total NKA quantity in diabetic nephropathy, the redistribution to the cystosol suggests the Na pump is no longer functional. ANGII also caused translocation fromthe basolateral membrane, thus in diabetic states where ANGII level is acutely elevated, the loss of NKA will be exacerbated. This provides another mechanism by which ANGII blockade is likely to be protective.
thank you for responding to my question. I have viewed that paper and thought that it was well done and have used this before as a reference. however, it does not help with my question about the molecular weight of the phospho NKA ser 23. I have run western blots in the presence of ang II from in vivo rat kidney cortex samples using an antibody from cell signaling. The data sheet for the antibody states that the band should be at 100 kDa. However, when I run the blot, I get 2 bands: 1 band at 65-70 and another band at 120-130 kDa. Both bands follow the predicted pattern that I would expect to have happened. For example, ang II caused an increase in phospho NKA ser 23 in both bands as published before. I am curious if in vivo samples the protein experiences post translational modifications or dimers and that is why I am seeing two bands. Any thoughts?
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