Detection of PHLPP1α/β in Human and Mouse Brain by Different Anti-PHLPP1 Antibodies

Travis C. Jackson; Hülya Bayir; Milos D. Ikonomovic; Keri Janesko-Feldman; Zaichuan Mi; Tianyan Gao; Edwin K. Jackson; Patrick M. Kochanek

doi:10.1038/srep09377

Detection of PHLPP1α/β in Human and Mouse Brain by Different Anti-PHLPP1 Antibodies

Travis C. Jackson
, Hülya Bayir
, Milos D. Ikonomovic
, Keri Janesko-Feldman
, Zaichuan Mi
, Tianyan Gao
, Edwin K. Jackson
, Patrick M. Kochanek

Research output: Contribution to journal › Article › peer-review

5 Scopus citations

Abstract

Pleckstrin homology domain and leucine rich repeat protein phosphatase 1 (PHLPP1) is a member of the serine/threonine family of phosphatases. It has been studied in organs including brain, heart, pancreas, adipose, breast, and prostate. Human PHLPP1 encodes two splice variants - PHLPP1α (∼140-150 kDa) and PHLPP1β (∼180-190 kDa). Commercial antibodies are widely used to characterize PHLPP1 proteins in cells/tissues. Here we validate five different antibodies to detect PHLPP1α/β by Western blot using PHLPP1 WT/KO mice. All antibodies recognize PHLPP1β in brain. Only a single antibody (Cosmo Bio Co) detects PHLPP1α (∼145-150 kDa). The other four antibodies detect a non-specific signal at ∼150 kDa as evidenced by its abundance in PHLPP1 KO tissues. Results suggest Cosmo antibody is a better reagent to detect PHLPP1α by Western blot. In contrast, we found it unsuitable for immunofluorescence applications in brain. Our findings caution interpretation of the ∼150 kDa band detected by some PHLPP1 antibodies in rodent and human tissues. Results also recapitulate the importance of including molecular weight standards in Western blot data to simplify retrospective analysis.

Original language	English
Article number	9377
Journal	Scientific Reports
Volume	5
DOIs	https://doi.org/10.1038/srep09377
State	Published - Apr 1 2015

Bibliographical note

Publisher Copyright:
© 2015, Nature Publishing Group. All rights reserved.

Funding

This work was supported in part by an American Heart Association grant to Travis C. Jackson (14SDG20210000), a U.S. army grant to Patrick M. Kochanek (Award# W81XWH-10-1-0623), and NIH grant R01CA133429 to Tianyan Gao. We would also like to thank and acknowledge the University of Pittsburgh ADRC which banked the human samples (NIH grant NIA AG05133 University of Pittsburgh ADRC).

Funders	Funder number
NIA AG05133 University of Pittsburgh ADRC
National Institutes of Health (NIH)
National Childhood Cancer Registry – National Cancer Institute	R01CA133429
American the American Heart Association	14SDG20210000, W81XWH-10-1-0623
University of Pittsburgh Medical Center, Children's Hospital of Pittsburgh

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title = "Detection of PHLPP1α/β in Human and Mouse Brain by Different Anti-PHLPP1 Antibodies",

abstract = "Pleckstrin homology domain and leucine rich repeat protein phosphatase 1 (PHLPP1) is a member of the serine/threonine family of phosphatases. It has been studied in organs including brain, heart, pancreas, adipose, breast, and prostate. Human PHLPP1 encodes two splice variants - PHLPP1α (∼140-150 kDa) and PHLPP1β (∼180-190 kDa). Commercial antibodies are widely used to characterize PHLPP1 proteins in cells/tissues. Here we validate five different antibodies to detect PHLPP1α/β by Western blot using PHLPP1 WT/KO mice. All antibodies recognize PHLPP1β in brain. Only a single antibody (Cosmo Bio Co) detects PHLPP1α (∼145-150 kDa). The other four antibodies detect a non-specific signal at ∼150 kDa as evidenced by its abundance in PHLPP1 KO tissues. Results suggest Cosmo antibody is a better reagent to detect PHLPP1α by Western blot. In contrast, we found it unsuitable for immunofluorescence applications in brain. Our findings caution interpretation of the ∼150 kDa band detected by some PHLPP1 antibodies in rodent and human tissues. Results also recapitulate the importance of including molecular weight standards in Western blot data to simplify retrospective analysis.",

author = "Jackson, \{Travis C.\} and H{\"u}lya Bayir and Ikonomovic, \{Milos D.\} and Keri Janesko-Feldman and Zaichuan Mi and Tianyan Gao and Jackson, \{Edwin K.\} and Kochanek, \{Patrick M.\}",

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AU - Bayir, Hülya

AU - Ikonomovic, Milos D.

AU - Janesko-Feldman, Keri

AU - Mi, Zaichuan

AU - Gao, Tianyan

AU - Jackson, Edwin K.

AU - Kochanek, Patrick M.

PY - 2015/4/1

Y1 - 2015/4/1

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Detection of PHLPP1α/β in Human and Mouse Brain by Different Anti-PHLPP1 Antibodies

Abstract

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