focal adhesion kinase fak inhibitor y15  (MedChemExpress)

Journal: Cell reports

Article Title: The mitotic ATR-Chk1 pathway promotes CDK1 activity for faithful chromosome segregation

doi: 10.1016/j.celrep.2025.116019

Figure Lengend Snippet: (A) The total number of phosphopeptides and the proportion of preferred consensus motifs for selected kinases that were downregulated (2-fold, p value<0.05) following 1-h treatment of ATR inhibitor (VE-821, ATRi) or Chk1 inhibitor (MK-8776, Chk1i) in phosphoproteomics screening of nocodazole-arrested and isolated U2OS cells. (B) Western blot quantification of the abundance of p-CDK1 Y15 over total CDK1 in 4-h nocodazole-arrested and isolated RPE-1 (left) and U2OS (right) cells that were either treated with vehicle control (DMSO), ATRi (VE-821), or Chk1i (MK-8776) for 1 h. (C) Western blot quantification of the abundance of p-CDK1 Y15 over total CDK1 in nocodazole-arrested wild-type (WT) A549 or heterozygous Chk1-knockout A549 cells (Chk1 +/− ) that were treated with either DMSO or Chk1i (CHIR-124) for 1 h. (D) Western blot quantification of the abundance of p-CDK1 Y15 over total CDK1 level in Taxol-arrested U2OS cells treated with either or Chk1i (CHIR-124) for 1 h. (E) Representative image (right) and quantification (left) of p-CDK1 T14 abundance over total CDK1 from immunofluorescence imaging of prometaphase U2OS cells from asynchronous population treated with either DMSO or Chk1i (MK-8776) for 20 min. (F) Representative image (right) and quantification (left) of p-CDK1 Y15 level from immunofluorescence imaging of prometaphase U2OS cells treated as in (E). (G) Representative image (right) and quantification (left) of p-PRC1 T481 from immunofluorescence imaging of prometaphase U2OS cells treated as in (E). For all western blot quantifications (B-D), the fold change for each replicate was normalized to the DMSO-treated control, n = 3–5 biological replicates, shown by data points on each graph. For immunofluorescence quantification (E-G), n > 150 cells over three biological replicates. Error bars in all panels represent SD. * p ≤ 0.05, by one-way ANOVA with a mixed-effects model of replicate averages (B), one-way ANOVA with Tukey’s correction (C), or Student’s t test (D-G) comparing replicate averages. Scale bars = 10 μm. See also .

Article Snippet: Rabbit anti- p -CDK1 Y15 , Bethyl Laboratories , Cat #A700-101; RRID: AB_2891898.

Techniques: Phospho-proteomics, Isolation, Western Blot, Control, Knock-Out, Immunofluorescence, Imaging

Journal: Cell reports

Article Title: The mitotic ATR-Chk1 pathway promotes CDK1 activity for faithful chromosome segregation

doi: 10.1016/j.celrep.2025.116019

Figure Lengend Snippet: (A) Western blot quantification of total Myt1 abundance in nocodazole-arrested U2OS cells that were treated with DMSO or Chk1i (CHIR-124) for 1 h. (B) p-Myt1 S83 abundance over total Myt1 levels in nocodazole-arrested U2OS cells treated as in (A). (C) Western blot quantification of p-CDK1 Y15 level over total CDK1 level in nocodazole-arrested U2OS cells treated with DMSO, Chk1i (CHIR-124), or combination of Chk1i and Wee1i (Wee1 inhibitor II) for 1 h. (D) Western blot quantification of p-CDK1 Y15 abundance over total CDK1 level in nocodazole-arrested U2OS cells treated with DMSO, Chk1i (CHIR-124), or Chk1i and Myt1i (RP-6306) for 1 h. (E) Western blot quantification of p-CDK1 Y15 level over total CDK1 level in U2OS cells treated with Myt1 siRNA for 48 h and subsequently arrested in nocodazole prior to isolation and treatment for 1 h with DMSO or Chk1i (CHIR-124). (F) Western blot quantification of p-CDK1 Y15 level over total CDK1 level in an in vitro kinase assay performed with untagged Chk1 (Chk1), Myc-DDK Myt1 (Myt1), and His-cyclinB1-CDK1 (CDK1). Myt1 was pre-incubated with Chk1 or heat-inactivated Chk1 (HI Chk1) for 1 h prior to the addition of heat-inactivated CDK1 for 20 min. For all western blot quantifications (A-F), the fold change for each replicate was normalized to the DMSO-treated control, n = 2–3 biological replicates, shown by data points on each graph. Error bars in all panels represent SD. * p ≤ 0.05 by Student’s t test (A-C) or one-way ANOVA with Tukey’s correction (D-F) of replicate averages. Scale bars = 10 μm. See also .

Article Snippet: Rabbit anti- p -CDK1 Y15 , Bethyl Laboratories , Cat #A700-101; RRID: AB_2891898.

Techniques: Western Blot, Isolation, In Vitro, Kinase Assay, Incubation, Control

Journal: Cell reports

Article Title: The mitotic ATR-Chk1 pathway promotes CDK1 activity for faithful chromosome segregation

doi: 10.1016/j.celrep.2025.116019

Figure Lengend Snippet: (A) Domain map of full-length Myt1 showing its kinase domain (KD), transmembrane domain (TM), and cyclin B-binding domain. The peptide identified using LC-MS/MS is shown below, depicting the Chk1 consensus sequence and phosphorylation at residue Serine 143. (B) Western blot of in vitro kinase assay of recombinant untagged Chk1 and Myc-DDK-Myt1 that were untreated or treated with lambda-phosphatase. (C) Representative immunofluorescence image (left) and quantification (right) of p-Myt1 S143-specific antibody staining in mitotic U2OS cells from asynchronous population treated with either DMSO or Chk1i (CHIR-124) for 20 min. Each data point for each replicate was divided by the mean integrated density of the DMSO-treated control. (D) Western blot quantification and comparison of the kinase activity of the full-length Myt1 (Myt1-FL) and truncated wild-type (WT) Myt1-KD using ADP-Glo assay. Either Myt1-FL or Myt1-KD was incubated with GST-tagged recombinant CDK1 for 30 min before the reaction was stopped for ADP-Glo assay. (E) Western blot quantification of p-CDK1 Y15 level over total CDK1 level in in vitro kinase assay where WT, kinase-dead (N238A), or phospho-null (S143A) mutations of the Myt1 KD were incubated with GST-CDK1 for 30 min. (F) Western blot quantification of p-CDK1 Y15 level over total CDK1 level in in vitro kinase assay with the WT Myt1-KD or phospho-null Myt1 KD (S143A Myt1-KD). WT Myt1-KD or S143A Myt1-KD were pre-incubated with Chk1 or heat-inactivated Chk1 (HI Chk1) for 1 h prior to the addition of GST-CDK1 for 60 min. Data points for (D-F) represent biological replicates. The mean of each replicate was normalized to Myt1-FL in (E), WT Myt1-KD in (F), and timepoint 0 in (G). For immunofluorescence quantification in (C). Statistical testing was performed on the mean integrated densities for each replicate. Error bars in all panels represent SD. * p ≤ 0.05 by Student’s t test (C, D, F) or one-way ANOVA with Tukey’s correction (E) for replicate averages. Scale bars = 10 μm. See also .

Article Snippet: Rabbit anti- p -CDK1 Y15 , Bethyl Laboratories , Cat #A700-101; RRID: AB_2891898.

Techniques: Binding Assay, Liquid Chromatography with Mass Spectroscopy, Sequencing, Phospho-proteomics, Residue, Western Blot, In Vitro, Kinase Assay, Recombinant, Immunofluorescence, Staining, Control, Comparison, Activity Assay, Glo Assay, Incubation

Journal: Cell reports

Article Title: The mitotic ATR-Chk1 pathway promotes CDK1 activity for faithful chromosome segregation

doi: 10.1016/j.celrep.2025.116019

Figure Lengend Snippet: (A) Representative immunofluorescence image (top) and intensity profile for Chk1, Myt1, and DNA of an untreated interphase U2OS cell. (B) Representative immunofluorescence image (top) and intensity profile (bottom) of an untreated mitotic U2OS cell. (C) Representative image (top) and intensity profile for p-Myt1 S143, tubulin, and DNA of an untreated interphase U2OS cell. (D) Quantification of the mean nuclear intensity of p-Myt1 S143 in untreated asynchronous U2OS cells in interphase or mitosis. Each data point for each replicate was divided by the mean of the DMSO-treated control. n = 3 biological replicates. (E) Representative immunofluorescence image (top) and intensity profile (bottom) of asynchronous U2OS cells transiently transfected with GFP-NLS-Myt1. (F) Western blot quantification of p-CDK1 T14 level over total CDK1 level in asynchronous U2OS population expressing either GFP-NLS-Myt1, GFP-NLS ΔTM Myt1, or none (untransfected, UNT). (G) Representative immunofluorescence image (top) and intensity profile (bottom) of asynchronous U2OS cells transiently transfected with GFP-NLS ΔTM Myt1. (H) Western blot quantification of p-CDK1 Y15 level over total CDK1 level in unstransfected U2OS (left) or transiently expressing GFP-NLS-Myt1 or GFP-NLS ΔTM Myt1 (right) treated with hydroxyurea (HU) for 24 h alone or HU for 24 h with 4 h of Chk1i treatment (HU + Chk1i). For (A-C), (E), and (F), the yellow arrow depicts the direction and length of the line used for the intensity profile. For all western blot quantifications, the fold change for each replicate was normalized to the DMSO-treated control. Data points represent biological replicates for panels (G-H). Error bars in all panels represent SD. * p ≤ 0.05 by Student’s t test (D, H) or one-way ANOVA with Tukey’s correction (F) for replicate averages. Scale bars = 10 μm. See also .

Article Snippet: Rabbit anti- p -CDK1 Y15 , Bethyl Laboratories , Cat #A700-101; RRID: AB_2891898.

Techniques: Immunofluorescence, Control, Transfection, Western Blot, Expressing

Journal: Cell reports

Article Title: The mitotic ATR-Chk1 pathway promotes CDK1 activity for faithful chromosome segregation

doi: 10.1016/j.celrep.2025.116019

Figure Lengend Snippet: (A) Percentage of mitotic U2OS cells undergoing anaphase with lagging chromosome after treatment with either DMSO, Chk1i (CHIR-124), or 0.5 μM CDK1i (RO-3306). (B) Percentage of mitotic HeLa cells undergoing anaphase with lagging chromosomes treated as in (A). (C) Representative images of mitotic U2OS anaphase cells treated with Chk1i. (D) Immunofluorescence quantification of p-H3 S28 (left) and p-H3 S10 (right) of U2OS cells that were treated with STLC for 4 h and subsequently treated with DMSO, Chk1i (CHIR-124), or low-dose CDK1i (RO-3306) for 1 h. Representative images of quantifications are shown on the right. (E) Quantification (left) and representative image (right) of the total p-INCENP T59 intensity along the chromosome in U2OS cells treated as in (A). Scale bars = 5 μm. (F) Schematic of our working model for the rewired Chk1-CDK1 pathway in mitosis. The ATR-Chk1 pathway inhibits Myt1 activity through Chk1’s direct phosphorylation of S143 on Myt1, decreasing its kinase activity and consequently decreasing p-CDK1 T14 and p-CDK1 Y15 levels on CDK1. This results in increased CDK1 activity, which promotes faithful chromosome segregation through the Aurora B kinase and the error correction machinery. For all the western blot quantifications, the fold change for each replicate was normalized to the DMSO-treated control. For immunofluorescence imaging quantifications, each data point for each replicate was divided by the mean integrated density of the DMSO-treated control. Statistical testing was performed on the mean integrated densities for each replicate. n = 3 biological replicates for all experiments. Error bars in all panels represent SD. *p ≤ 0.05 by one-way ANOVA with Tukey’s correction (B-D). Scale bars = 10 μm, unless indicated otherwise. See also .

Article Snippet: Rabbit anti- p -CDK1 Y15 , Bethyl Laboratories , Cat #A700-101; RRID: AB_2891898.

Techniques: Immunofluorescence, Activity Assay, Phospho-proteomics, Western Blot, Control, Imaging