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.
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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.
Jackson, Travis C.; Bayir, Hülya; Ikonomovic, Milos D.; Janesko-Feldman, Keri; Mi, Zaichuan; Gao, Tianyan; Jackson, Edwin K.; and Kochanek, Patrick M., "Detection of PHLPP1α/β in Human and Mouse Brain by Different Anti-PHLPP1 Antibodies" (2015). Markey Cancer Center Faculty Publications. 35.
Figure 1: PHLPP1 Antibody Comparison in WT and KO Brain.
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Figure 2: Utility of Cosmo Antibody for Western and Immunofluorescence Applications.
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Figure 3: Detection of PHLPP1 Variants in Human Neuronal cells and Brain Tissue.
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Figure 4: PHLPP1 Antibody Comparison in Peripheral Organs from WT and KO Mice.