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. 2011 Feb 15;286(14):12766–12774. doi: 10.1074/jbc.M111.224014

FIGURE 1.

FIGURE 1.

Characterization hPER2 phospho-FASPS cluster antibodies. A, sequence overlay between mammalian CREB and PER proteins. Homologous putative phosphorylation sites are shown in bold and underlined. Putative ATM phosphorylation sites in the PER proteins are highlighted in yellow. The residue Ser-662, mutated in FASPS, is italicized and marked with an asterisk. B, phosphatase sensitivity of pPER2(FASPS) antisera. HEK 293T cells were transfected with vector DNA (−) or a plasmid encoding Myc epitope-tagged hPER2 for 24 h. The cell extracts were prepared and treated with λ phosphatase prior to analysis by SDS-PAGE and immunoblotting with α-Myc and α-pPER2(FASPS) antibodies. C, phosphorylation site requirements. HEK 293T cells were transfected with plasmids encoding Myc-tagged hPER2WT or the indicated hPER2 phosphorylation site mutants. The cell extracts were then prepared and analyzed by immunoblotting using α-Myc and α-pPER2(FASPS) antibodies. D, α-pPER2(FASPS) antibodies are selective for hPER2. HEK 293T cells were transfected with plasmids encoding Myc-tagged hPER2WT or the hPER2A664V mutant. The cell extracts were then made and analyzed by immunoblotting using α-Myc and α-pPER2(FASPS) antibodies.