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.
authors.affiliation: University of Pittsburgh School of Medicine, Department of Critical Care Medicine, Safar Center for Resuscitation Research, 200 Hill Building, 3434 Fifth Avenue.
authors.forename: Travis C
authors.initials: TC
authors.lastname: Jackson
authors.affiliation: University of Pittsburgh School of Medicine, Department of Critical Care Medicine, Safar Center for Resuscitation Research, 200 Hill Building, 3434 Fifth Avenue.
authors.forename: Hülya
authors.initials: H
authors.lastname: Bayir
authors.affiliation: University of Pittsburgh School of Medicine, Department of Neurology, 811 Kaufmann Medical Building, 3471 Fifth Avenue.
authors.forename: Milos D
authors.initials: MD
authors.lastname: Ikonomovic
authors.affiliation: University of Pittsburgh School of Medicine, Department of Critical Care Medicine, Safar Center for Resuscitation Research, 200 Hill Building, 3434 Fifth Avenue.
authors.forename: Keri
authors.initials: K
authors.lastname: Janesko-Feldman
authors.affiliation: University of Pittsburgh School of Medicine, Department of Pharmacology and Chemical Biology, Bridgeside Point Building 1, 100 Technology Drive.
authors.forename: Zaichuan
authors.initials: Z
authors.lastname: Mi
authors.affiliation: University of Kentucky College of Medicine, Department of Molecular &Cellular Biochemistry, Markey Cancer Center, Lexington, KY 40536, USA.
authors.forename: Tianyan
authors.initials: T
authors.lastname: Gao
authors.affiliation: University of Pittsburgh School of Medicine, Department of Pharmacology and Chemical Biology, Bridgeside Point Building 1, 100 Technology Drive.
authors.forename: Edwin K
authors.initials: EK
authors.lastname: Jackson
authors.affiliation: University of Pittsburgh School of Medicine, Department of Critical Care Medicine, Safar Center for Resuscitation Research, 200 Hill Building, 3434 Fifth Avenue.
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Kochanek
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism*; D005260:Female; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms
chemical_list: D009687:Nuclear Proteins; D020033:Protein Isoforms; C400601:PHLPP1 protein, human; C519282:PHLPP1 protein, mouse; D010749:Phosphoprotein Phosphatases
references.citation: J Neurosci. 2013 Nov 27;33(48):18880-92
references.pmid: 24285894
references.citation: J Pharmacol Exp Ther. 2013 Nov;347(2):516-28
references.pmid: 24023368
references.citation: J Biol Chem. 2009 Sep 18;284(38):25813-22
references.pmid: 19546214
references.citation: Proc Natl Acad Sci U S A. 2010 Jan 26;107(4):1642-7
references.pmid: 20080691
references.citation: Biotechniques. 2010 Mar;48(3):197-209
references.pmid: 20359301
references.citation: J Biol Chem. 2003 Apr 25;278(17):14920-5
references.pmid: 12594205
references.citation: FEBS Lett. 1999 Sep 24;458(3):363-9
references.pmid: 10570941
references.citation: J Neurochem. 2010 Nov;115(4):941-55
references.pmid: 20819118
references.citation: Mol Cell. 2005 Apr 1;18(1):13-24
references.pmid: 15808505
references.citation: J Comp Neurol. 2005 Dec 26;493(4):477-8
references.pmid: 16304632
references.citation: Cell. 2007 Mar 23;128(6):1219-29
references.pmid: 17382888
references.citation: Mol Cell. 2007 Mar 23;25(6):917-31
references.pmid: 17386267
references.citation: J Histochem Cytochem. 2009 Jan;57(1):1-5
references.pmid: 18854594
references.citation: Nat Struct Mol Biol. 2011 Jan;18(1):91-3
references.pmid: 21131980
references.citation: EMBO Rep. 2013 Jan;14(1):10-1
references.pmid: 23229585
references.citation: J Cereb Blood Flow Metab. 2013 Feb;33(2):196-204
references.pmid: 23072745
references.citation: EMBO Rep. 2013 Jun;14(6):494-6
references.pmid: 23681440
references.citation: Cell Death Differ. 2009 Mar;16(3):439-48
references.pmid: 19039330