chk2 activator  (Santa Cruz Biotechnology)

Journal: Cell Death & Disease

Article Title: ELK3 destabilization by speckle-type POZ protein suppresses prostate cancer progression and docetaxel resistance

doi: 10.1038/s41419-024-06647-0

Figure Lengend Snippet: a Dephosphorylation of ELK3 prevent the interaction between ELK3 and SPOP. The cell lysates of HEK293T cells transiently transfected with indicated plasmids were treated with/without λ-phosphatase. The interaction between ELK3 and SPOP was evaluated by IP and WB. b CHK1/2 inhibition suppresses ELK3 destabilization. The cell lysates of HeLa cells treated with 5 μM of indicated inhibitor and CHX (10 μg/ml) for 12 h were used to evaluate the ELK3 protein levels by WB. c CHK1/2 inhibition abolishes ELK3 and SPOP interaction. The cell lysates of HEK293T cells transfected with indicated plasmids treated with 5 μM of indicated inhibitor for 12 h and MG132 (10 μM) for 4 h were used to evaluate the interaction between ELK3 and SPOP by IP and WB. d ELK3 interacts with CHK1 and CHK2. The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate the interaction between ELK3 and CHK1 or CHK2 by IP and WB. e CHK2 facilitates ELK3 ubiquitination by SPOP. cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate the ELK3 ubiquitination by IP and WB. f CHK1/2 inhibition abrogates SPOP-mediated ELK3 ubiquitination. The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated with 5 μM of AZD7762 for 12 h and MG132 (10 μM) for 4 h were used to evaluate the SPOP-mediated ELK3 ubiquitination by IP and WB. g CHK1 knockdown increases ELK3 protein levels. The cell lysates of HeLa cells stably expressing sh-mock or sh-CHK1 or CHK2 were used to evaluate ELK3 protein levels by WB. h CHK1 and CHK2 phosphorylates ELK3. In vitro kinase assay using partially purified His-ELK3 and active CHK1 or CHK2 was conducted. ELK3 phosphorylation by CHK1 or CHK2 was evaluated RxxS/T antibody by WB. i ELK3 deg1 deletion abolishes CHK1- or CHK2-mediated phosphorylation. In vitro kinase assay using partial purified His-ELK3-wt or -∆Deg1 and active CHK1 or CHK2 was conducted. ELK3 phosphorylation by CHK1 or CHK2 was evaluated RxxS/T antibody by WB. j , k CHK2 phosphorylates ELK3 at Ser133 in cell system. The phosphorylation of ELK3 mediated by CHK2 in cell culture ( j ) and in vitro kinase assay system ( k ) was evaluated using phos-tag immunoblot analysis. Treatment with AZD7762 ( j ) and mutation of ELK3 Ser133 to Ala ( l ) abolished the phosphorylation of ELK3 induced by CHK2. l ELK3 phosphorylation at Ser133 is indispensable to interact with SPOP. ELK3 phosphorylation requirement for the interaction with SPOP was evaluated by IP and western blotting using cell lysates transiently expressing mock, His-ELK3-wt, His-ELK3-S133D, or His-ELK3-S133A.

Article Snippet: Subsequently, the partially purified ELK3-wt (1 μg) and ELK3-ΔDeg1 (1 μg) were combined with active CHK1 (cat. no.: C47-10H, SignalChem, Richmond, BC, Canada) and CHK2 (cat. no.: C48-10G, SignalChem) and cold ATP.

Techniques: De-Phosphorylation Assay, Transfection, Inhibition, Ubiquitin Proteomics, Knockdown, Stable Transfection, Expressing, In Vitro, Kinase Assay, Purification, Phospho-proteomics, Cell Culture, Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: Aberrant subcellular localization of mismatch repair protein MLH1 dysregulates the cell cycle to create new therapeutic opportunities

doi: 10.1101/2024.02.27.582389

Figure Lengend Snippet: (A) Schema showing association between cytoplasmic MLH1 that results into impaired Chk2 activation and its effect on CDK activity. (B) RT-qPCR analysis of MCF7 cells for relative expression of indicated transcripts following fulvestrant treatment. (C) Bar graph showing growth of MCF7 cells with G55V or WT MLH1 constructs after siCDK6 knockdown alone or with fulvestrant treatment. Associated data validating knockdowns and growth in T47D cells in Fig S8 A, B. (D) Bar graph showing growth of MCF7 cells with G55V or WT MLH1 constructs after CDK4/6 inhibitor palbociclib treatment. Associated data for T47D cells in Fig S8C. (E) 3D growth of T47D cells harboring G55V or WT MLH1 after palbociclib treatment. (F) In vivo tumor growth expressed as change in tumor volume of MCF7 cells stably expressing G55V mutant and WT constructs after palbociclib treatment normalized to vehicle. Associated data showing tumor growth in vehicle and palbociclib is presented in Fig S8 F-G. (G) Bar graph showing effect of palbociclib treatment in three PDX derived organoids (PDoX) models. 3D cell-titre glo was performed to measure viability. Growth is expressed relative to vehicle (ultrapure water treatment) for each PDoX. (H-I) CDK4/6 inhibitor response expressed as percent Treatment/ Control ratio in PDX lines with cytoplasmic or nuclear MLH1 (H) Palbociclib (I) Abemaciclib. Student’s t test was performed to determine all p values. For panel H and I Fisher’s exact test was performed to calculate p values. *** represents p<0.0001, ** p<0.005 and * p<0.05. For all graphs, error bars describe standard deviation.

Article Snippet: CHK2 activator (3, 3;-diindolyl methane, cat# sc-204624; Santa Cruz Biotechnology) was used at 100 μmol/L concentrations.

Techniques: Activation Assay, Activity Assay, Quantitative RT-PCR, Expressing, Construct, Knockdown, In Vivo, Stable Transfection, Mutagenesis, Derivative Assay, Control, Standard Deviation

Journal: Cell reports

Article Title: Epinephrine inhibits PI3Kα via the Hippo kinases

doi: 10.1016/j.celrep.2023.113535

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CHK2 , SignalChem , C48–10G.

Techniques: Recombinant, Fluorescence, Isolation, Double Knockout, Mutagenesis, Plasmid Preparation, Software