mca1360 rrid ab 322378 a phospho cdc2 tyr15 cell signaling technology  (Cell Signaling Technology Inc)

Journal: International Journal of Oncology

Article Title: Ricolinostat enhances adavosertib-induced mitotic catastrophe in TP53-mutated head and neck squamous cell carcinoma cells

doi: 10.3892/ijo.2022.5344

Figure Lengend Snippet: Ricolinostat suppresses phosphorylation of Chk1 and further suppresses p-CDK1 when co-administered with adavosertib. (A) CAL27 cells and (B) TP53-WT and TP53-KO A549 cells were treated with Adv (0.5 µ M), RCS (5 µ M), and Adv+RCS for 24 h (CAL27 cells) or 48 h (A549 cells), and then the expression of DNA damage response-related proteins (p-Chk2, Chk2, p-ATR, ATR, p-Chk1, Chk1, p-CDK1, CDK1, and γ-H2A.X) was assessed by western blotting. To assess the inhibitory effect of HDAC6 by RCS, the level of acetylated (ac)-α-tubulin was monitored. Expression of β-actin was assessed as the loading control. The relative band intensity of each phosphorylated protein was calculated and summarized at the right. Representative data of three independent experiments are shown. Adv, adavosertib; RCS, ricolinostat; WT, wild-type; Chk, checkpoint kinase; ATR, ATR serine/threonine kinase; CDK1, cyclin-dependent kinase 1.

Article Snippet: These membranes were probed with primary antibodies, such as anti-p53 antibody (Ab) (sc-126, 1/1,000), anti-β-actin Ab (sc-47778, 1/1,000), anti-HDAC6 Ab (sc-11420, 1/1,000), anti-acetylated α-tubulin Ab (sc-23950, 1/1,000), and anti-α-tubulin Ab (sc-5286, 1/1,000) were purchased from Santa Cruz Biotechnology, Inc. Anti-PARP Ab (#9542S, 1/1,000), anti-caspase3 Ab (#9665S, 1/1,000), anti-phospho-p53 Ab (#9286, 1/1,000), anti-p21 Ab (#2947S, 1/1,000), anti-phospho-ATR Ab (#9947, 1/1,000), anti-ATR Ab (#2790, 1/1,000), anti-phospho-Chk1 (Ser345) Ab (#2348, 1/1,000), anti-Chk1 Ab (#2360, 1/1,000), anti-phospho-Chk2 (Thr68) Ab (#2197, 1/1,000), anti-Chk2 Ab (#3440, 1/1,000), anti-phospho-Cdc2 (Tyr15) Ab (#4539, 1/1,000), anti-Cdc2 Ab (#9116, 1/1,000), anti-H2A.X Ab (#7631, 1/1,000), and anti-phospho-histone H2A.X (Ser139) Ab (#9718, 1/1,000) were purchased from Cell Signaling Technology, Inc.

Techniques: Phospho-proteomics, Expressing, Western Blot, Control

Journal: International Journal of Oncology

Article Title: Ricolinostat enhances adavosertib-induced mitotic catastrophe in TP53-mutated head and neck squamous cell carcinoma cells

doi: 10.3892/ijo.2022.5344

Figure Lengend Snippet: Schema of the effect of adavosertib and ricolinostat on the G2/M checkpoint. In TP53 -mutated cells, DNA repair is dependent on the G2/M checkpoint regulated by CDK1. CDK1 is a key regulator inducing cell cycle progression through the G2/M checkpoint. WEE1 phosphorylates and inhibits CDK1. Adavosertib inhibits WEE1 and then CDK1 is dephosphorylated and activated. Chk1 inhibits Cdc25, which dephosphorylates and activates CDK1. Ricolinostat decreases the phosphorylation of Chk1, indicating inhibition of Chk1. Chk, checkpoint kinase; ATR, ATR serine/threonine kinase; ATM, ATM serine/threonine kinase; CDK1, cyclin-dependent kinase 1; HDAC6, histone deacetylase 6; WEE1, WEE1 G2 checkpoint kinase.

Article Snippet: These membranes were probed with primary antibodies, such as anti-p53 antibody (Ab) (sc-126, 1/1,000), anti-β-actin Ab (sc-47778, 1/1,000), anti-HDAC6 Ab (sc-11420, 1/1,000), anti-acetylated α-tubulin Ab (sc-23950, 1/1,000), and anti-α-tubulin Ab (sc-5286, 1/1,000) were purchased from Santa Cruz Biotechnology, Inc. Anti-PARP Ab (#9542S, 1/1,000), anti-caspase3 Ab (#9665S, 1/1,000), anti-phospho-p53 Ab (#9286, 1/1,000), anti-p21 Ab (#2947S, 1/1,000), anti-phospho-ATR Ab (#9947, 1/1,000), anti-ATR Ab (#2790, 1/1,000), anti-phospho-Chk1 (Ser345) Ab (#2348, 1/1,000), anti-Chk1 Ab (#2360, 1/1,000), anti-phospho-Chk2 (Thr68) Ab (#2197, 1/1,000), anti-Chk2 Ab (#3440, 1/1,000), anti-phospho-Cdc2 (Tyr15) Ab (#4539, 1/1,000), anti-Cdc2 Ab (#9116, 1/1,000), anti-H2A.X Ab (#7631, 1/1,000), and anti-phospho-histone H2A.X (Ser139) Ab (#9718, 1/1,000) were purchased from Cell Signaling Technology, Inc.

Techniques: Phospho-proteomics, Inhibition, Histone Deacetylase Assay

Journal: Cell reports

Article Title: MMB-FOXM1-driven premature mitosis is required for CHK1 inhibitor sensitivity

doi: 10.1016/j.celrep.2021.108808

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: pY15 CDK1/2 , Cell Signaling Technologies , Cat# 9111S; RRID: AB_331460.

Techniques: Genome Wide, Recombinant

Journal: Cell reports

Article Title: MMB-FOXM1-driven premature mitosis is required for CHK1 inhibitor sensitivity

doi: 10.1016/j.celrep.2021.108808

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: pT161 CDK1 , Cell Signaling Technologies , Cat# 9114S; RRID: AB_2074652.

Techniques: Genome Wide, Recombinant

Journal: Cell reports

Article Title: The Apparent Requirement for Protein Synthesis during G2 Phase Is due to Checkpoint Activation

doi: 10.1016/j.celrep.2020.107901

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: rabbit anti-Cdk1 phospho-Tyr15 , Cell Signaling Technology , #9111L; RRID:AB_331460.

Techniques: Recombinant, Software

Journal: Molecular cell

Article Title: Histone chaperones ASF1 and CAF-1 promote MMS22L-TONSL-mediated Rad51 loading onto ssDNA during homologous recombination in human cells

doi: 10.1016/j.molcel.2018.01.031

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Phospho-cdc2 (Tyr15) , Cell Signaling Technology , Cat# 9111S; RRID: AB_10835695.

Techniques: Recombinant, Transfection, Lysis, Bradford Protein Assay, Western Blot, SYBR Green Assay, shRNA, Plasmid Preparation, Software