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+/HCO3− 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.
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References
Mochly-Rosen D, Basbaum AI, Koshland DE Jr (1987) Distinct cellular and regional localization of immunoreactive protein kinase C in rat brain. Proc Natl Acad Sci U S A 84(4):4660–4664
Wetsel WC, Khan WA, Merchenthaler I, Rivera H, Halpern AE, Phung HM, Negro-Vilar A, Hannun YA (1992) Tissue and cellular distribution of the extended family of protein kinase C isoenzymes. J Cell Biol 117(1):121–133
Shi SR, Key ME, Kalra KL (1991) Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 39(4):741–748
Ohno N, Terada N, Murata S, Katoh R, Ohno S (2005) Application of cryotechniques with freeze-substitution for the immunohistochemical demonstration of intranuclear pCREB and chromosome territory. J Histochem Cytochem 53(1):55–62
Saitoh S, Terada N, Ohno N, Ohno S (2008) Distribution of immunoglobulin-producing cells in immunized mouse spleens revealed with “in vivo cryotechnique”. J Immunol Methods 331(1–2):114–126
Terada N, Ohno N, Saitoh S, Ohno S (2007) Immunohistochemical detection of hypoxia in mouse liver tissues treated with pimonidazole using “in vivo cryotechnique”. Histochem Cell Biol 128(3):253–261
Miller RT (2005) Phospho-specific antibodies: more valuable than originally thought. Am J Physiol Renal Physiol 289(2):247–248
He H, Tan M, Pamarthy D, Wang G, Ahmed K, Sun Y (2007) CK2 phosphorylation of SAG at Thr10 regulates SAG stability, but not its E3 ligase activity. Mol Cell Biochem 295(1–2):179–188
Pearson RB, Kemp BE (1991) Protein kinase phosphorylation site sequences and consensus specificity motifs: tabulations. Methods Enzymol 200:62–81
Nishizuka Y (1988) The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature 334(6148):661–665
Bonventre JV (1993) Mechanisms of ischemic acute renal failure. Kidney Int 43(5):1160–1178
Sakai N, Sasaki K, Ikegaki N, Shirai Y, Ono Y, Saito N (1997) Direct visualization of the translocation of the gamma-subspecies of protein kinase C in living cells using fusion proteins with green fluorescent protein. J Cell Biol 139(6):1465–1476
Miyawaki A, Llopis J, Heim R, McCaffery JM, Adams JA, Ikura M, Tsien RY (1997) Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388(6645):882–887
Ng T, Squire A, Hansra G, Bornancin F, Prevostel C, Hanby A, Harris W, Barnes D, Schmidt S, Mellor H, Bastiaens PI, Parker PJ (1999) Imaging protein kinase Calpha activation in cells. Science 283(5410):2085–2089
Saitoh S, Terada N, Ohno N, Saitoh Y, Soleimani M, Ohno S (2009) Immunolocalization of phospho-Arg-directed protein kinase-substrate in hypoxic kidneys using in vivo cryotechnique. Med Mol Morphol (42):24–31
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Saitoh, S., Terada, N., Ohno, N., Saitoh, Y., Ohno, S. (2016). Immunolocalization of Phospho-Arg-Directed Protein Kinase-Substrate in Hypoxic Kidneys. In: Ohno, S., Ohno, N., Terada, N. (eds) In Vivo Cryotechnique in Biomedical Research and Application for Bioimaging of Living Animal Organs. Springer, Tokyo. https://doi.org/10.1007/978-4-431-55723-4_25
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DOI: https://doi.org/10.1007/978-4-431-55723-4_25
Publisher Name: Springer, Tokyo
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