ck1γ1  (Carna Inc)

Journal: eLife

Article Title: Cdc7 activates replication checkpoint by phosphorylating the Chk1-binding domain of Claspin in human cells

doi: 10.7554/eLife.50796

Figure Lengend Snippet: ( A ) C-terminally Flag-tagged wild type (WT) and mutant Claspin indicated were transiently expressed in 293 T cells and were pulled down by anti-Flag beads. Immunoprecipitated proteins were analyzed by western blotting with anti-Flag and anti-Chk1 antibodies. Shown below the panels are schematic diagrams of the mutant Claspin proteins. Black boxes indicated the segments in which amino acid replacements were introduced. Asterisks indicate IgG; arrowheads indicate Chk1. WT(HA) represents the HA-tagged Claspin, which was used as a negative control. ( B ) Stable clones of Claspin flox /-MEF cells expressing the Flag-tagged wild-type or AP DE/A mutant Claspin or no transgene were infected with Ad-Cre for 48 hr or non-treated. Cells were then treated with CK1γ1 siRNA (lanes 7–12) or non-treated (lanes 1–6) for 24 hr and then with 2 mM HU for 3 hr, and the whole cell extracts were analyzed by western blotting with indicated antibodies. ( C ) U2OS cells were infected by retroviruses expressing mAG-WT Claspin or mAG-AP DE/A Claspin (momeric Azami Green fluorescent protein fused at the N-terminus of Claspin, which does not affect the functionso of Clapsin). At 2 days after infection, non-coding Claspin siRNA or control siRNA was introduced into the cells for two days, and then the cells were treated with 2 mM HU for 1 hr or non-treated before harvest. The whole cell extracts were analyzed by western blotting with indicated antibodies. mAG indicates monomeric Azami Green fluorescent protein.

Article Snippet: Purified Cdc7-ASK (05–109), Chk1 (02–117) and CK1γ1(03–105) kinase were from Carna Bioscience, Inc Cdc7-ASK was also purified from Sf9 cells as previously described ( ).

Techniques: Mutagenesis, Immunoprecipitation, Western Blot, Negative Control, Clone Assay, Expressing, Infection

Journal: eLife

Article Title: Cdc7 activates replication checkpoint by phosphorylating the Chk1-binding domain of Claspin in human cells

doi: 10.7554/eLife.50796

Figure Lengend Snippet: ( A ) Purified Claspin polypeptide #27 (aa897-1100 containing CKBD and AP) was incubated in the kinase reaction with indicated amounts of purified Cdc7-ASK and/or CK1γ1 in the presence of [γ- 32 P]-ATP. Samples were analyzed on 4–20% gradient SDS-PAGE, followed by silver staining (right) and autoradiogram (left). The positions of the proteins are indicated by arrows. Bottom drawing shows schematic diagram of the polypeptide #27. ( B ) Quantification of the phosphorylation level of the #27 polypeptide in A .

Article Snippet: Purified Cdc7-ASK (05–109), Chk1 (02–117) and CK1γ1(03–105) kinase were from Carna Bioscience, Inc Cdc7-ASK was also purified from Sf9 cells as previously described ( ).

Techniques: Purification, Incubation, SDS Page, Silver Staining

Journal: eLife

Article Title: Cdc7 activates replication checkpoint by phosphorylating the Chk1-binding domain of Claspin in human cells

doi: 10.7554/eLife.50796

Figure Lengend Snippet: ( A ) Two hundred ng (34 pmole) each of wild type CKBD peptide (wild-type) or mutant peptides (CKBD-A, Others-A and Others-E) was incubated in the kinase reaction with indicated amounts of purified Cdc7-ASK and/or CK1γ1 in the presence of [γ- 32 P]-ATP for 60 min at 30°C. Samples were analyzed on 18% SDS-PAGE. After silver staining, the gel was autoradiographed. The position of the substrate peptides in the gel is indicated by an arrow. The amino acid sequences of each peptide are shown below the panel. The serine and threonine in CKBD are in green, and the mutated residues are shown in red. ( B ) Quantification of the phosphorylation level of the peptide in ( A ). The averages of three independent kinase assays are shown with error bars. Figure 6—source data 1. Quantification for graph (three independent FACS experiments) in .

Article Snippet: Purified Cdc7-ASK (05–109), Chk1 (02–117) and CK1γ1(03–105) kinase were from Carna Bioscience, Inc Cdc7-ASK was also purified from Sf9 cells as previously described ( ).

Techniques: Mutagenesis, Incubation, Purification, SDS Page, Silver Staining

Journal: eLife

Article Title: Cdc7 activates replication checkpoint by phosphorylating the Chk1-binding domain of Claspin in human cells

doi: 10.7554/eLife.50796

Figure Lengend Snippet: The whole cell extracts from 5 × 10 4 293T, HeLa, HCT116, U2OS and NHDF cells and purified Cdc7 and CK1γ1 proteins of the amount indicated were run on 4–20% gradient SDS-PAGE and analyzed by western blotting with antibodies against Cdc7 (upper) or CK1γ1 (lower). The band intensities were quantified and the numbers of Cdc7 or CK1γ1 molecules per cell were calculated. The averages of the three experiments with standard deviations are indicated in the table on the right.

Article Snippet: Purified Cdc7-ASK (05–109), Chk1 (02–117) and CK1γ1(03–105) kinase were from Carna Bioscience, Inc Cdc7-ASK was also purified from Sf9 cells as previously described ( ).

Techniques: Purification, SDS Page, Western Blot

Journal: eLife

Article Title: Cdc7 activates replication checkpoint by phosphorylating the Chk1-binding domain of Claspin in human cells

doi: 10.7554/eLife.50796

Figure Lengend Snippet: ( A ) NHDF cells were infected by Cdc7- or CK1γ1-expressing lentiviruses. At 2 days after infection, cells were selected by puromycin (4 µg/ml) for 2 days, followed by incubation in the absence of the selection for 3 weeks. Cells were then transfected with indicated siRNA for 2 days, followed by incubation with 2 mM HU for 50 min. The whole cell extracts were analyzed by western blotting with indicated antibodies. ( B ) Quantification of the levels of phosphorylation Chk1 in ( A ).

Article Snippet: Purified Cdc7-ASK (05–109), Chk1 (02–117) and CK1γ1(03–105) kinase were from Carna Bioscience, Inc Cdc7-ASK was also purified from Sf9 cells as previously described ( ).

Techniques: Infection, Expressing, Incubation, Selection, Transfection, Western Blot

Journal: eLife

Article Title: Cdc7 activates replication checkpoint by phosphorylating the Chk1-binding domain of Claspin in human cells

doi: 10.7554/eLife.50796

Figure Lengend Snippet: ( A ) Purified wild type Claspin or Claspin mutant ST27A (serines and threonines in aa903-1120 substituted by alanine) was incubated in the kinase reaction with indicated amounts of purified CK1γ1 in the presence of [γ- 32 P]-ATP. Samples were analyzed on 4–20% gradient SDS-PAGE, followed by CBB staining (right) and autoradiogram (left). The positions of the proteins are indicated by arrows. Bottom drawing shows schematic diagram of the polypeptide #27. ( B ) Quantification of the phosphorylation levels of the Wt or ST27A Claspin in A . The background phosphorylation level in the absence of CK1γ1 was taken as one for both WT and ST27A Claspin proteins.

Article Snippet: Purified Cdc7-ASK (05–109), Chk1 (02–117) and CK1γ1(03–105) kinase were from Carna Bioscience, Inc Cdc7-ASK was also purified from Sf9 cells as previously described ( ).

Techniques: Purification, Mutagenesis, Incubation, SDS Page, Staining