title: Immunolocalization of phospho-Arg-directed protein kinase-substrate in hypoxic kidneys using in vivo cryotechnique.
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 cryofixed with IVCT under normal blood circulation and after varying hypoxic intervals, followed by freeze-substitution with acetone containing paraformaldehyde. Deparaffinized 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 identified the same 40-kDa bands. The IVCT is useful for time-dependent analysis of the immunodistribution of P-PK-S and P-Akt-S.
authors.affiliation: Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi, 409-3898, Japan.
authors.forename: Sei
authors.initials: S
authors.lastname: Saitoh
authors.forename: Nobuo
authors.initials: N
authors.lastname: Terada
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Ohno
authors.forename: Yurika
authors.initials: Y
authors.lastname: Saitoh
authors.forename: Manoocher
authors.initials: M
authors.lastname: Soleimani
authors.forename: Shinichi
authors.initials: S
authors.lastname: Ohno
mesh_terms: D000818:Animals; D001120:Arginine / Q000378:metabolism; D015925:Cryopreservation; D003593:Cytoplasm / Q000201:enzymology; D017110:Freeze Substitution; D000860:Hypoxia / Q000201:enzymology* / Q000473:pathology; D015151:Immunoblotting; D007150:Immunohistochemistry; D007668:Kidney / Q000201:enzymology; D007674:Kidney Diseases / Q000201:enzymology* / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001120:Arginine; D011494:Protein Kinases; D051057:Proto-Oncogene Proteins c-akt