vitro phosphorylated mbp hd fusion protein  (Millipore)

Journal: Molecular and Cellular Biology

Article Title: Identification of the In Vivo Casein Kinase II Phosphorylation Site within the Homeodomain of the Cardiac Tisue-Specifying Homeobox Gene Product Csx/Nkx2.5

doi:

Figure Lengend Snippet: Csx/Nkx2.5 phosphorylation increases DNA binding affinity. Csx/Nkx2.5 protein expressed in COS cells was dephosphorylated with CIAP, and DNA binding activity was compared to that of phosphorylated Csx/Nkx2.5. (A) Transfected-cell lysates were separated by SDS-PAGE and blotted with Csx/Nkx2.5 MAb. The majority of Csx/Nkx2.5 protein was dephosphorylated by CIAP treatment (lane 2). A 5-μl aliquot of each cell lysate (lanes 1 and 2) and 5 ng of MBP-Csx/Nkx2.5 fusion protein contained equivalent amounts of Csx/Nkx2.5 protein. (B) EMSAs with ANF Csx/Nkx2.5 binding site (TGAAGTG). The DNA-Csx/Nkx2.5 protein complex (arrowhead) was supershifted by anti-Csx/Nkx2.5 MAb (arrow) in a dose-dependent manner. ANF probe was end-labeled in this experiment. (C) EMSA with ANF Csx/Nkx2.5 binding site. Lanes CIAP+ contain twofold serially diluted CIAP-treated Csx/Nkx2.5 protein; lanes CIAP− contain untreated Csx/Nkx2.5 protein. The shifted bands were scanned and plotted against protein concentration (lower panel). The plotted line was shifted downward by CIAP treatment, indicating phosphorylated Csx/Nkx2.5 has higher DNA binding affinity than dephosphorylated protein. (D) DNA mobility shift assay (similar to that shown in panel C) using A20 Csx/Nkx2.5 binding site (AGTTAATTG). Phosphorylated protein (lanes CIAP−) showed higher DNA binding affinity than dephosphorylated protein.

Article Snippet: Immunoprecipitants of in vivo-labeled Csx/Nkx2.5 or in vitro-phosphorylated MBP-HD fusion protein were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore) after SDS-PAGE and then autoradiographed.

Techniques: Binding Assay, Activity Assay, Transfection, SDS Page, Labeling, Protein Concentration, Mobility Shift

Journal: Molecular and Cellular Biology

Article Title: Identification of the In Vivo Casein Kinase II Phosphorylation Site within the Homeodomain of the Cardiac Tisue-Specifying Homeobox Gene Product Csx/Nkx2.5

doi:

Figure Lengend Snippet: Csx/Nkx2.5 is phosphorylated in vivo and in vitro. (A) COS cells transfected with Csx/Nkx2.5 expression vector (lanes 1 and 3) or pcDNA3 vector alone (lanes 2 and 4) were 32 P labeled, lysed, and immunoprecipitated (IP) with Csx/Nkx2.5 MAb (lanes 1 and 2) or control IgG1 (lanes 3 and 4). The 37-kDa Csx/Nkx2.5 band was specifically immunoprecipitated with Csx/Nkx2.5 MAb (lane 1) but not with control IgG1 (lane 3). (B) MBP alone (MBP), MBP-homeodomain Csx/Nkx2.5 (HD), and MBP-full length Csx/Nkx2.5 (Csx) were phosphorylated with [γ- 32 P]ATP and 2 μg of cytoplasmic (Cyt) or nuclear (Nuc) extract of NIH 3T3 cells. Samples were subjected to SDS-PAGE, and phosphorylated proteins were visualized by autoradiography. Both cytoplasmic (lanes 1 to 3) and nuclear lysates (lanes 4 to 6) approximately equally phosphorylated Csx/Nkx2.5 proteins (lane 2 versus lane 5; lane 3 versus lane 6). MBP was not phosphorylated by either cytoplasmic (lane 1) or nuclear (lane 4) extracts.

Article Snippet: Immunoprecipitants of in vivo-labeled Csx/Nkx2.5 or in vitro-phosphorylated MBP-HD fusion protein were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore) after SDS-PAGE and then autoradiographed.

Techniques: In Vivo, In Vitro, Transfection, Expressing, Plasmid Preparation, Labeling, Immunoprecipitation, SDS Page, Autoradiography

Journal: Molecular and Cellular Biology

Article Title: Identification of the In Vivo Casein Kinase II Phosphorylation Site within the Homeodomain of the Cardiac Tisue-Specifying Homeobox Gene Product Csx/Nkx2.5

doi:

Figure Lengend Snippet: Phosphorylation of Csx/Nkx2.5 by CKII within the homeodomain. (A) In-gel kinase assays. NIH 3T3 cell nuclear extracts (25 μg) were separated in a gel containing either the homeodomain-MBP fusion protein (HD), the full-length Csx/Nkx2.5-MBP fusion protein (Csx), or MBP. After denaturation and renaturation, gels were incubated in kinase buffer containing [γ- 32 P]ATP. One kinase with a molecular mass of ∼40 kDa (arrowheads) phosphorylated the Csx/Nkx2.5 homeodomain, Csx/Nkx2.5 (lane 1), and the full-length Csx/Nkx2.5 (lane 2), but not MBP (lane 3). (B) Schematic representation of Csx/Nkx2.5 mutants carrying the consensus CKII sites and serine-to-alanine substitutions. (C) In vitro CKII phosphorylation of Csx/Nkx2.5. Three different Csx/Nkx2.5 fusion proteins described in panel B were incubated with [γ- 32 P]ATP in the presence (+) or absence (−) of purified CKII. Each sample was subjected to SDS-PAGE and transferred to a PVDF membrane. Autoradiography (upper panel) revealed that CKII phosphorylated the full-length Csx/Nkx2.5 (lane 1) and the homeodomain (lane 3) but neither the C-terminally deleted mutant (lane 5) nor MBP (lane 7). Loaded fusion proteins are shown by Western blotting of the same PVDF membrane using anti-MBP antibody (lower panel). (D) Serine 163 was mutated into alanine (Csx/Nkx2.5 163S-A ) and the kinase assay was performed. Lane 1 contains the wild-type Csx/Nkx2.5 and lane 2 contains the mutant Csx/Nkx2.5 163S-A . Equal amounts of proteins were subjected to the kinase reaction as shown in the MBP Western blot (lower panel).

Article Snippet: Immunoprecipitants of in vivo-labeled Csx/Nkx2.5 or in vitro-phosphorylated MBP-HD fusion protein were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore) after SDS-PAGE and then autoradiographed.

Techniques: Incubation, In Vitro, Purification, SDS Page, Autoradiography, Mutagenesis, Western Blot, Kinase Assay

Journal: Molecular and Cellular Biology

Article Title: Identification of the In Vivo Casein Kinase II Phosphorylation Site within the Homeodomain of the Cardiac Tisue-Specifying Homeobox Gene Product Csx/Nkx2.5

doi:

Figure Lengend Snippet: CKII phosphorylation increases DNA binding affinity. (A) EMSA with ANF Csx/Nkx2.5 binding site. Csx/Nkx2.5 homeodomain fusion protein (MBP-HD) (1 ng) incubated with either CKII (lanes CKII+) or heat-inactivated CKII (lanes CKII−) and their twofold serially diluted proteins were assayed for DNA binding. The shifted bands were scanned and plotted against the protein concentration (lower panel). CKII treatment shifted the plotted line upward, indicating that CKII-phosphorylated protein has higher DNA binding affinity. Slower-migrating bands might represent the dimerized protein. The first lane contains the gel shift with non-fused MBP protein; the last lane contains the free probe without added fusion protein. (B) A result similar to that shown in panel A is observed with A20 Csx/Nkx2.5 binding site.

Article Snippet: Immunoprecipitants of in vivo-labeled Csx/Nkx2.5 or in vitro-phosphorylated MBP-HD fusion protein were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore) after SDS-PAGE and then autoradiographed.

Techniques: Binding Assay, Incubation, Protein Concentration, Electrophoretic Mobility Shift Assay

Journal: Molecular and Cellular Biology

Article Title: Identification of the In Vivo Casein Kinase II Phosphorylation Site within the Homeodomain of the Cardiac Tisue-Specifying Homeobox Gene Product Csx/Nkx2.5

doi:

Figure Lengend Snippet: Phosphopeptide and phosphoamino acid analysis of Csx/Nkx2.5. (A) Tryptic phosphopeptide mapping of wild-type Csx/Nkx2.5 (a), CKII mutant (b), and in vitro CKII-phosphorylated MBP-homeodomain protein (c). Metabolically 32 P-labeled Csx/Nkx2.5 and the CKII mutant (Csx/Nkx2.5 163S-A ) were immunoprecipitated with anti-Csx/Nkx2.5 MAb (2D10), resolved by SDS-PAGE, transferred to a PVDF membrane, and autoradiographed. In vitro CKII-phosphorylated MBP-homeodomain protein was also electrophoresed and autoradiographed. These protein bands were cut out, digested with trypsin, and resolved on TLC plates by electrophoresis in the first dimension and chromatography in the second dimension. In vitro CKII-phosphorylated peptide (c [arrow]) which corresponded to peptide 1 in the wild type (a [arrow]), was markedly reduced in the CKII mutant (b). Arrowheads, sample loaded points; X, lysine marker. (B) Phosphoamino acid analysis of wild-type Csx/Nkx2.5 showed Csx/Nkx2.5 is phosphorylated predominantly in S and weakly in T.

Article Snippet: Immunoprecipitants of in vivo-labeled Csx/Nkx2.5 or in vitro-phosphorylated MBP-HD fusion protein were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore) after SDS-PAGE and then autoradiographed.

Techniques: Phosphoamino Acid Analysis, Mutagenesis, In Vitro, Metabolic Labelling, Labeling, Immunoprecipitation, SDS Page, Thin Layer Chromatography, Electrophoresis, Chromatography, Marker