Quantitative analysis of phosphorylated AQP2 in urine exosomes

Urine exosome samples were denatured in SDS sample-buffer (Nacalai Tesque, Kyoto, Japan) for 20 min at 75 °C. Then, the samples were separated by SDS-PAGE and the proteins were transferred to a PVDF membrane (Immobilon-P, Merk KGaA, Darmstadt, Germany). The blots were probed with following primary antibodies: rabbit anti AQP2 antibody for total AQP2 [5 (link)], rabbit anti pS256-AQP2 antibody (Abcam, Cambridge, UK) [13 (link), 14 (link)], rabbit anti pS261-AQP2 antibody (PhosphoSolutions, Aurora, USA) [14 (link)], rabbit anti pS264-AQP2 antibody (PhosphoSolutions) [15 (link)] and rabbit anti pS269-AQP2 antibody (PhosphoSolutions) [14 (link)]. Alkaline-phosphatase-conjugated anti rabbit IgG antibody (Promega, Madison, USA) was used as a secondary antibody and Western blue (Promega) was used to detect the signals [13 (link)]. The band intensities of the western blots were quantified using ImageJ software (https://imagej.nih.gov/ij/).

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Sakai M., Yamamoto K., Mizumura H., Matsumoto T., Tanaka Y., Noda Y., Ishibashi K., Yamamoto T, & Sasaki S. (2020). Phosphorylation profile of human AQP2 in urinary exosomes by LC–MS/MS phosphoproteomic analysis. Clinical and Experimental Nephrology, 24(9), 762-769.

Publication 2020

SDS sample-buffer Western blue

Alkaline phosphatase Anti antibody Antibodies Antibody Buffer Exosome Igg antibody Immobilon p Membrane Promega Proteins Pvdf Rabbit Sds page Urine Western blots

Corresponding Organization : Tokyo Medical and Dental University

Other organizations : Nakano General Hospital

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Total AQP2 protein levels
Phosphorylation levels of AQP2 at residues S256, S261, S264, and S269

control variables

Denaturation of urine exosome samples in SDS sample-buffer for 20 min at 75 °C
SDS-PAGE separation of proteins
Transfer of proteins to a PVDF membrane
Use of primary antibodies: rabbit anti AQP2, rabbit anti pS256-AQP2, rabbit anti pS261-AQP2, rabbit anti pS264-AQP2, and rabbit anti pS269-AQP2
Use of alkaline-phosphatase-conjugated anti rabbit IgG antibody as secondary antibody
Use of Western blue to detect signals
Quantification of band intensities using ImageJ software

positive controls

None explicitly mentioned

negative controls

None explicitly mentioned

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