Journal: Cell Cycle

Article Title: Identification of the proteome complement of humanTLK1 reveals it binds and phosphorylates NEK1 regulating its activity

doi: 10.1080/15384101.2017.1314421

Figure Lengend Snippet: (A) The activation of ATR (autophosphorylation at T1989) after 6h incubation with hydroxyurea (HU) is strongly reduced by concomitant addition of the TLK1 inhibitor, thioridazine (THD, 10 µM). (B) Activation of ATR (pATR-T1989) is impaired following 1h incubation with 0.2 mM H2O2 in cells expressing NEK1-T141A in contrast to wt-NEK1 expressing cells. Where indicated, siRNA to nek1 (si1) was added 24h before H2O2. (C) Activation of Chk1 (pChk1-S345) is reduced in cells overexpressing NEK1-T141A.

Article Snippet: Antibodies used in this study were: rabbit α-Actin (Ab1801, Abcam), rabbit α-Chk1 phospho-317 (AP3070a, Abgent), rabbit α-Chk1 phospho-345 (sc-17922, Santa Cruz Biotechnology), rabbit α-TLK1 (GTX102891, GeneTex), rabbit αNEK1 Antibody, A304–570A (Bethyl Lab), mouse monoclonal α Nek1 (E-10) (sc-398813 Santa Cruz Biotechnology), mouse monoclonal Anti-polyHistidine antibody (H1029, SIGMA), rabbit αATR antibody (A300–138A, Bethyl Lab), rabbit αpATR (phospho Thr1989) antibody (GTX128145, GeneTex), donkey α-goat IgG-HRP (sc-2020, Santa Cruz Biotechnology), α-rabbit IgG-HRP (7074S, Cell Signaling), α-mouse IgG-HRP (7076, Cell Signaling), mouse α- H2A.X phospho-139 (05–636, Millipore).

Techniques: Activation Assay, Incubation, Expressing

Journal:

Article Title: Phosphorylated hMSH6: DNA mismatch versus DNA damage recognition

doi: 10.1016/j.mrfmmm.2010.10.008

Figure Lengend Snippet: HeLa MR cells were synchronized by DTB and released into media with or without 2 µM MNNG. At 12 h, cells were further incubated with or without 200 nM UCN-01. Cells were harvested at indicated times for cross-linked chromatin, cytoplasmic extract, or flow cytometry. A. Equal amounts of cross-linked chromatin were subjected to SDS-PAGE, transferred to PVDF membrane and immunoblotted with antibody to phosphorylated Chk1 (S345 and S317). Equal amounts of HeLa MR cytoplasmic extract were subjected to SDS-PAGE, transferred to PVDF membrane and immunoblotted with antibodies to phosphorylated Cdc25c (S216) and GAPDH (loading control), or phosphorylated Cdk1 (T15) and total Cdk1. B. HeLa MR cells were harvested at indicated time points to monitor cell cycle progression by flow cytometric analysis of DNA content.

Article Snippet: 2.1 Materials Monoclonal antibodies against hMSH6 (BD Biosciences), hMSH2 (Calbiochem), phospho-serine (Calbiochem), Chk1 (Santa Cruz Biotechnology), phospho-Chk1 Ser 345 and Ser 317 (Cell Signaling), phospho-Cdc25c Ser 216 (Santa Cruz Biotechnology), Cdc2 (Cdk1) (Cell Signaling), and phospho-Cdc2 (pCdk1) Tyr 15 (Cell Signaling) were purchased from the indicated suppliers.

Techniques: Incubation, Flow Cytometry, SDS Page

Journal:

Article Title: Erbb2 Suppresses DNA Damage-Induced Checkpoint Activation and UV-Induced Mouse Skin Tumorigenesis

doi: 10.2353/ajpath.2009.080638

Figure Lengend Snippet: Erbb2 blocks Chk1 activation through a PI3K/Akt dependent mechanism after UV irradiation. Keratinocytes in culture were treated with 45 μmol/L AG825 or the vehicle DMSO (A–D, F) or Erbb2fl/fl keratinocytes were infected with Cre recombinase expressing or empty virus (D) and sham-irradiated or exposed to 600 J/m2 UV 2 hours (for inhibitor) or 24 hours later (for Cre recombinase infection). Protein lysate was obtained at the indicated time points after UV irradiation (A–D, F). A: ATR/M activity was determined by immunoblotting for ATR/M substrate phosphorylation using an antibody specific for the phosphorylated consensus ATR/M substrate sequence and immunoblotting for actin. The sum of the signal from all bands detected with the ATR/M substrate phosphorylation antibody was normalized to actin levels using densitometry. The mean and SE for at least six samples at each time point and treatment is shown. No significant differences were detected between DMSO and AG825 treated samples at any time point. B: Chk1 phosphorylation on Ser345 was determined after immunoblotting with a phospho-Chk1-Ser345 specific antibody (inset). The signal for Chk1 phosphorylation relative to actin was determined using densitometry and the results from two replicate experiments averaged and graphed. Mean ± SEM is shown. C: Immunoblotting for Chk1 and Chk2 phosphorylation on abrogation of Erbb2 and UV exposure in cell lysates (“P” = phospho). D: Akt activation, measured by Akt phosphorylation (P-AKT), is dependent on Erbb2 activation after UV irradiation. “m” indicates minutes post-UV. E: Inhibition of PI3K or Akt, as described in Materials and Methods, causes S-phase arrest 24 hours after UV irradiation. N ≥4 experiments. Significantly different when compared with the vehicle-treated and UV-irradiated control, where *P ≤ 0.05. F: Immunoblotting for inhibitory phosphorylation of Chk1 on Ser280 on abrogation of Erbb2 activity and UV irradiation.

Article Snippet: Membranes were immunoblotted with antibodies recognizing actin (Sigma), phosphorylated ATR/M substrates (Cell Signaling, Danvers, MA), Cdc25a (Santa Cruz Biotechnology, Santa Cruz, CA), Cdc25b (Cell Signaling), Cdc25c (Santa Cruz Biotechnology), phospho-Chk1-Ser 296 (Cell Signaling), phospho-Chk1-Ser 280 (gift of Ramon Parsons), phospho-Chk1 345 (Santa Cruz), phospho-Chk2 387 (Cell Signaling), and phospho-Akt (Cell Signaling, Beverly, MA), horseradish peroxidase-conjugated secondary antibodies (Cell Signaling), and visualized using chemiluminescent reagents (Pierce, Rockford, IL).

Techniques: Activation Assay, Irradiation, Infection, Expressing, Activity Assay, Western Blot, Sequencing, Inhibition

Journal:

Article Title: Erbb2 Suppresses DNA Damage-Induced Checkpoint Activation and UV-Induced Mouse Skin Tumorigenesis

doi: 10.2353/ajpath.2009.080638

Figure Lengend Snippet: Erbb2 regulates cell cycle progression following UV exposure by suppressing ATR checkpoint activation. A: In normal cells, UV-induced Erbb2 activation in turn activates PI3K/Akt signaling, which suppresses Chk1 activation and subsequent degradation of Cdc25a. B: On abrogation of Erbb2 activity, increased Chk1/2 activity leads to decreased Cdc25a and subsequently, to increased S-phase arrest. The net effect of decreased Erbb2 activity is decreased skin tumor development, presumably because of improved DNA repair and decreased mutagenesis.

Article Snippet: Membranes were immunoblotted with antibodies recognizing actin (Sigma), phosphorylated ATR/M substrates (Cell Signaling, Danvers, MA), Cdc25a (Santa Cruz Biotechnology, Santa Cruz, CA), Cdc25b (Cell Signaling), Cdc25c (Santa Cruz Biotechnology), phospho-Chk1-Ser 296 (Cell Signaling), phospho-Chk1-Ser 280 (gift of Ramon Parsons), phospho-Chk1 345 (Santa Cruz), phospho-Chk2 387 (Cell Signaling), and phospho-Akt (Cell Signaling, Beverly, MA), horseradish peroxidase-conjugated secondary antibodies (Cell Signaling), and visualized using chemiluminescent reagents (Pierce, Rockford, IL).

Techniques: Activation Assay, Activity Assay, Mutagenesis