Research ResourceBiochemistry

Antibodies recognizing the C terminus of PP2A catalytic subunit are unsuitable for evaluating PP2A activity and holoenzyme composition

See allHide authors and affiliations

Science Signaling  28 Jan 2020:
Vol. 13, Issue 616, eaax6490
DOI: 10.1126/scisignal.aax6490
  • Fig. 1 Antibodies raised against the C terminus of PP2A C preferentially recognize the nonmethylated C subunit of PP2A.

    (A) Posttranslational modifications of the PP2A C-terminal tail and schematic of PP2A holoenzyme maturation. The PP2A core enzyme consists of a structural A subunit and a catalytic C subunit. LCMT-1 methylates PP2A C. The methylated PP2A core enzyme associates with B, B′, or B″ regulatory subunits. The unmethylated core enzyme associates with striatin, SV40 ST, PyST, or MT regulatory subunits. (B) Quantification of monoclonal antibody 1D6 binding to the peptides Leu309 (Ac-HVTRRTPDYFL) and meLeu309 (Ac-HVTRRTPDYFL-Me) by ELISA. Antibody binding data are shown as the average and SD of n = 4 independent ELISA experiments. The signals were normalized to Leu309 peptide, which was arbitrarily set to 1. (C) Immunoblotting of lysates from untreated or NaOH-treated HAP1 (HAP) and HEK293Trex (HEK) cells using the indicated antibodies. The panel originates from two different blotting membranes used for the C subunit antibodies (1D6 and H8). The 1D6 blot was incubated with a pan-actin antibody as a loading control. The blots are representative of n = 5 (HAP) or n = 3 (HEK) independent immunoblotting experiments. The blots were quantified using a dilution series of the NaOH-treated sample (fig. S1B). (D) Immunoblotting of lysates from untreated or NaOH-treated HAP1 cells using the indicated antibodies. The panel originates from seven different blotting membranes used for the C subunit–specific antibodies. The H8 blot was reincubated with pan-actin antibody as a loading control. The blots are representative of n = 3 or n = 4 independent immunoblotting experiments. The blots were quantified using a dilution series of the NaOH-treated sample and normalized to the H8 signal. Statistical significance of ELISA and immunoblotting quantifications were assessed using Student’s t test. *P < 0.05, **P < 0.01, ****P < 0.001; a.u., arbitrary units.

  • Fig. 2 Antibodies raised against the C terminus of PP2A C weakly immunoprecipitate B or B′ holoenzymes and cross-react with PP4 C.

    (A) Immunoblotting of HA, 1D6, 7A9, and IgG (immunoglobulin G; control) immunoprecipitates (IP) and lysates (1/10 of input) from HEK293Trex cells transfected with either empty vector (control) or vector encoding HA-PP2A C subunit using antibodies H8, 1D7, or 7C10 or antibodies directed against the indicated A and B subunits. The panels originate from three different blotting membranes. The blots are representative of n = 3 independent immunoprecipitation experiments. The A subunit and striatin coimmunoprecipitations were quantified from three independent experiments relative to the amount of immunoprecipitated PP2A C, and the HA-PP2A C IP was arbitrarily set to 1. (B) Quantification of the catalytic activity of HA, 1D6, or 2G9 immunoprecipitates from lysates of HEK293Trex cells transfected with either vector encoding HA-PP2A C subunit (for HA-PP2A immunoprecipitation) or control vector (for 1D6 and 2G9 immunoprecipitations) analyzed by phosphatase assays toward phosphorylase a or CDK1-phosphorylated histone H1. The measured activity was normalized to the amount of immunoprecipitated PP2A C subunit, and the specific activity of HA-PP2A C IP was set to 100%. n = 4 independent immunoprecipitation and phosphatase assay experiments. (C) Alignment of the C termini of mammalian C subunits of PP2A, PP4, and PP6. (D) Immunoblotting of HA immunoprecipitates from lysates of NIH3T3 cells infected with either retroviral supernatants (control) or retrovirus carrying HA-PP2A C, HA-PP4 C, or HA-PP6 C using the indicated antibodies. To equilibrate HA amounts, two times more of the HA-PP4 and three times more of the HA-PP6 C immunoprecipitates were loaded compared with HA-PP2A. The panel originates from 11 different blotting membranes that are representative of n = 3 independent immunoprecipitation experiments. Statistical significance of immunoblotting and phosphatase assay quantifications was assessed using ANOVA followed by Tukey’s HSD as a post hoc test. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001; C, PP2A C subunit; B, PP2A B subunit.

  • Fig. 3 Methylation sensitivity is the source of a detection bias in the 1D6-based commercial immunoprecipitation phosphatase assay kit.

    (A) The catalytic activity of 1D6 immunoprecipitates from wild-type and Lcmt-1 HAP1 cells was analyzed with the PP2A immunoprecipitation phosphatase assay kit (Millipore). The activity of wild type was set to 100% and was calculated both without normalization to the immunoprecipitated PP2A C subunit and with normalization to the immunoprecipitated PP2A C subunit. n = 3 independent experiments. (B) 1D6 immunoprecipitates (IP) from the PP2A phosphatase activity kit on wild-type and Lcmt-1 HAP1 cells were immunoblotted using the indicated antibodies. The panels that are shown originate from the same blotting membrane and are representative of three independent experiments. Statistical significance of immunoblotting and phosphatase assay quantifications was assessed using Student’s t test. *P < 0.05.

  • Fig. 4 Analysis of 1D6 immunoprecipitates fails to detect the nearly complete absence of B-containing trimers in Lcmt-1 cells.

    (A) Lysates and 1D6 and 2G9 immunoprecipitates (IP) of wild-type or Lcmt-1 HAP1 cells were analyzed by immunoblotting with the indicated antibodies. IgG immunoprecipitates are the control. Blots that are shown originate from three different blotting membranes and are representative of n = 5 (1D6) or n = 3 (2G9) independent immunoprecipitation experiments. The PP2A C and PP4 C immunoprecipitation was quantified from n = 5 independent experiments, and the immunoprecipitations from wild-type cells were set to 1. (B) The catalytic activities of 1D6 and 2G9 immunoprecipitates from wild-type and Lcmt-1 HAP1 cells were analyzed by phosphatase activity toward histone H1. The measured activity was normalized to activity immunoprecipitated with 1D6 from wild-type cells, which was arbitrarily set to 100% and was calculated by either normalization to the immunoprecipitated PP2A C subunit or normalization to activity immunoprecipitated with 2G9 from wild-type cells without normalization to PP2A C abundance. Statistical significance of immunoblotting and phosphatase assay quantifications was assessed using Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.005; ND, not determined.

  • Table 1 Summary of results with C-terminal antibodies.

    This table shows an overview of the properties of the tested C-terminal PP2A C antibodies. For details, please see results and figures. ND, not determined

    CloneSensitive to meLeu309Sensitive to pThr304Sensitive to
    pTyr307
    Cross-reacts
    with PP4
    Cross-reacts
    with PP6
    Immunoprecipitates PP2A
    holoenymes
    1D6YesYesYesYesNoNo
    7A6YesYesYesYesNoNo
    G-4YesYesYesYesNoND
    52F8YesNDNDNoNoNo
    2038YesNDNDYesYesND
    Detects meLeu309Sensitive to pThr304Sensitive to
    pTyr307
    Cross-reacts
    with PP4
    Cross-reacts
    with PP6
    Immunoprecipitates PP2A
    holoenymes
    7C10YesNoNoNoNoNo
    2A10YesNDNDYesYesND
    1D7NoYesNoNoNoND

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/13/616/eaax6490/DC1

    Fig. S1. 1D6 preferentially recognizes nonmethylated PP2A C subunit.

    Fig. S2. Phosphorylation of Thr304 or Tyr307 influences epitope recognition by PP2A C-terminal antibodies.

    Fig. S3. 1D6 and other C-terminal antibodies do not immunoprecipitate two major PP2A holoenzyme families.

    Fig. S4. 7C10 specifically recognizes methylated PP2A C, and 2A10 also recognizes methylated PP4 C.

    Table S1. Antibodies used in this study.

  • This PDF file includes:

    • Fig. S1. 1D6 preferentially recognizes nonmethylated PP2A C subunit.
    • Fig. S2. Phosphorylation of Thr304 or Tyr307 influences epitope recognition by PP2A C-terminal antibodies.
    • Fig. S3. 1D6 and other C-terminal antibodies do not immunoprecipitate two major PP2A holoenzyme families.
    • Fig. S4. 7C10 specifically recognizes methylated PP2A C, and 2A10 also recognizes methylated PP4 C.
    • Table S1. Antibodies used in this study.

    [Download PDF]

Stay Connected to Science Signaling

Navigate This Article