Immunolocalization of phospho-arg-directed protein kinase-substratein hypoxic kidneys

Sei Saitoh, Nobuo Terada, Nobuhiko Ohno, Yurika Saitoh, Shinichi Ohno

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Protein kinases (PKs) phosphorylate proteins at active regions for signal transduction. In this study, normal and hypoxic mouse kidneys were prepared using an “in vivo cryotechnique” (IVCT) and examined immunohistochemically with specific antibodies against phospho-(Ser/Thr) PKA/C substrate (P-PK-S) and phospho-(Ser/Thr) Akt substrate (P-Akt-S) to capture their time-dependent regulation in vivo. Left kidneys were cryofi xed with IVCT under normal blood circulation and after varying hypoxic intervals, followed by freeze-substitution with acetone containing paraformaldehyde. Deparaffi nized sections were immunostained for P-PK-S, Na + /HCO 3− cotransporter NBC1, and a membrane skeletal protein, 4.1B. The P-PK-S was diffusely immunolocalized in the cytoplasm of the proximal tubules in normal kidneys, whereas NBC1 and 4.1B were detected at the basal striations of S1 and S2 segments of the proximal tubule. After 10 or 30 s hypoxia, P-PK-S was still immunolocalized in the cytoplasm of kidneys, but it was detected at the basal striations after 1 or 2 min hypoxia. The immunolocalization of P-Akt-S was the same as P-PK-S in the normal and hypoxic kidneys. Immunoblotting analyses of the kidney tissues under normal or hypoxic condition clearly identifi ed the same 40 kDa bands. The IVCT is useful for timedependent analysis of the immunodistribution of P-PK-S and P-Akt-S.

Original languageEnglish
Title of host publicationIn Vivo Cryotechnique in Biomedical Research and Application for Bioimaging of Living Animal Organs
PublisherSpringer Japan
Pages135-140
Number of pages6
ISBN (Electronic)9784431557234
ISBN (Print)9784431557227
DOIs
Publication statusPublished - 01-01-2015
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

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