ser 1981 p atm antibody  (R&D Systems)

Journal: EMBO Reports

Article Title: Tumor acidosis-induced DNA damage response and tetraploidy enhance sensitivity to ATM and ATR inhibitors

doi: 10.1038/s44319-024-00089-7

Figure Lengend Snippet: Reagents and tools.

Article Snippet: Antibodies targeted against phosphorylated p-ATM (D25E5) (Ser-1981) (1:1000), ATM (D2E2) (1:1000), p-ATR (D5K8W) (Thr-1989) (1:1000), ATR (E1S3S) (1:1000), p-CHK1 (133D3) (Ser-345) (1:1000), CHK1 (2G1D5) (1:1000), p-CHK2 (C13C1) (Thr-68) (1:1000), CHK2 (1C12) (1:1000) and α/β-tubulin (1:2000) are from Cell Signaling Technologies; β-Actin (AC-15) (1:30,000) from Sigma-Aldrich.

Techniques: Membrane, Modification, Protease Inhibitor, Software, Bicinchoninic Acid Protein Assay, Viability Assay

Journal: Oncotarget

Article Title: Camptothecin induces G 2 /M phase arrest through the ATM-Chk2-Cdc25C axis as a result of autophagy-induced cytoprotection: Implications of reactive oxygen species

doi: 10.18632/oncotarget.24934

Figure Lengend Snippet: ( A ) LNCaP cells were incubated with 4 μM CPT for the indicated time points. Cytosol and nuclear lysates were resolved on SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and probed with antibodies against Nrf2. ( B ) LNCaP cells were pretreated with NAC (5 mM) for 2 h and then incubated with CPT (4 μM) for indicated time points. Nuclear extracts were prepared to analyze ARE-binding of Nrf2 by EMSA. ( C ) Cells were transiently transfected with Nrf2 -targeted siRNA (siNrf2) or with control siRNA (siCON), and then Nrf2 expression was examined by western blot analysis. ( D ) At 12 h, representative histograms for the effect of CPT treatment on cell cycle distribution in LNCaP cells transfected with siNrf2 or with siCON ( top ). A parallel experiment was used to measure ROS formation by staining cells with H 2 DCFDA-based fluorescence under similar condition ( bottom ). ( E ) Effect of siRNA-based Nrf2 protein depletion on cell cycle proteins. LNCaP cells were incubated with 4 μM CPT for the indicated time. Cytosol and nuclear lysates were resolved on SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and probed with antibodies against p21, Cdk2, and cyclin B1. β-Actin was used as an internal control for western blot analysis. ( F ) Effect of siNrf2 on cell cycle distribution in the presence of the ROS inhibitors, NAC and GSH. LNCaP cells were transiently transfected with siNrf2 for 24 h and then treated with 5 mM NAC and 5 mM GSH for 1 h prior to incubation with 4 μM CPT for 12 h. Representative histograms for the effect of CPT treatment on cell cycle distribution as determined by flow cytometry.

Article Snippet: Antibodies against Cdk2, cyclin B1, p21, β-actin, Nrf2, nucleolin, phospho (p)-ATM (Ser 1981 ), ATM, Chk1, Chk2, p-Chk2 (Thr 68 ), Cdc25c, p-Cdc25C (Ser 216 ), ubiquitin, procaspase-3, procaspase-9, Cdk2, Bad, Beclin-1, LC3, and Atg-7 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Incubation, Binding Assay, Transfection, Control, Expressing, Western Blot, Staining, Fluorescence, Flow Cytometry

Journal: Oncotarget

Article Title: Camptothecin induces G 2 /M phase arrest through the ATM-Chk2-Cdc25C axis as a result of autophagy-induced cytoprotection: Implications of reactive oxygen species

doi: 10.18632/oncotarget.24934

Figure Lengend Snippet: LNCaP cells were cultured in the presence of 4 μM CPT for 24 h. ( A ) Western blot analysis of the effects of CPT on levels of phosphorylated ATM and Chk1/2. ( B – C ) Effect of the ATM inhibitor on CPT-induced Chk1/2 expression. LNCaP cells were pretreated with the ATM inhibitor prior to incubation with 4 μM CPT for 24 h. Total protein was subjected to 10% SDS-PAGE followed by western blot analysis with antibodies specific for phosphorylated forms of Chk1 and Chk2 (B). In a parallel experiment, the cells were harvested, stained with propidium iodide and analyzed for cell cycle distribution (C). ( D ) Transient knockdown of Chk1 and Chk2. Chk1 - and Chk2 -targeted siRNA (siChk1 and siChk2) were transiently transfected into LNCaP cells for 48 h and then the expression levels of Chk1 and Chk2 were analyzed by western blot analysis. ( E ) After LNCaP cells were transfected with siChk1 ( top ) and siChk2 ( bottom ) for 24 h and then treated with 4 μM CPT for additional 24 h. DNA content was analyzed by flow cytometry. ( F ) LNCaP cells were pretreated with NAC (5 mM) for 1 h and then incubated with CPT (4 μM). ATM phosphorylation and expression of Chk1 and Chk2 were measured by western blot analysis. β-Actin was used as an internal control.

Article Snippet: Antibodies against Cdk2, cyclin B1, p21, β-actin, Nrf2, nucleolin, phospho (p)-ATM (Ser 1981 ), ATM, Chk1, Chk2, p-Chk2 (Thr 68 ), Cdc25c, p-Cdc25C (Ser 216 ), ubiquitin, procaspase-3, procaspase-9, Cdk2, Bad, Beclin-1, LC3, and Atg-7 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Cell Culture, Western Blot, Expressing, Incubation, SDS Page, Staining, Knockdown, Transfection, Flow Cytometry, Phospho-proteomics, Control

Journal: Oncotarget

Article Title: Camptothecin induces G 2 /M phase arrest through the ATM-Chk2-Cdc25C axis as a result of autophagy-induced cytoprotection: Implications of reactive oxygen species

doi: 10.18632/oncotarget.24934

Figure Lengend Snippet: ( A ) Western blot analysis of Cdc25C levels using lysates from untreated control group and CPT-treated LNCaP cells. LNCaP cells were cultured in the presence of 4 μM CPT for 24 h. Total protein was subjected to 10% SDS-PAGE followed by western blotting with antibodies specific for phosphorylated forms of Cdc25C. ( B ) LNCaP cells were cultured in the presence of 4 μM CPT for 24 h. Cells were measured by dual analysis of p-Cdc25C and DNA content in control and CPT-treated cells. ( C ) Effect of Chk2 depletion on CPT-induced phosphorylation of Cdc25C and cell cycle protein. siChk2-transfected cells were treated with 4 μM CPT for 24 h, harvested and processed for western blot analysis using antibodies against Cdc25C, p-Cdc25C, cyclin B1, and p21. β-Actin was used as an internal control. ( D ) Effect of proteasome inhibitor MG132 on CPT-induced decline in Cdc25C protein levels. LNCaP cells were treated with 4 μM CPT in the presence or absence of MG132 for 24 h. Cell lysates prepared for western blot analysis using antibodies against ubiquitin to identify the high molecular weight polyubiquitin conjugates. ( E ) Effect of MG132 on CPT-induced cell cycle arrest. LNCaP and Hep3B cells were treated with 4 μM CPT in the presence or absence of MG132 prior to processing for analysis of cell cycle distribution. ( F ) Effect of treatment with combination of CPT and MG132 on DNA fragmentation. After treatment of LNCaP cells as indicated for 24 h, fragmented DNAs were extracted from the cells and analyzed on 1.5% agarose gels. ( G ) Cells with sub-G 1 phase DNA content were detected by flow cytometry. The percentages of cells with sub-G 1 DNA content are shown in each panel. ( H ) Effect of treatment with a combination of CPT and MG132 on levels of pro-apoptotic and anti-apoptotic protein. LNCaP cells were treated with 2 μM MG132 alone, 4 μM CPT alone, or a combination of both for 24 h. Cell extracts were prepared for western blot analysis for caspase-9, caspase-3, and Bad. β-Actin was used as an internal control. Data from three independent experiments are expressed as the overall mean ± S.E. Statistical significance was determined by one-way ANOVA ( a and b , p < 0.05 vs. control and CPT-treated group).

Article Snippet: Antibodies against Cdk2, cyclin B1, p21, β-actin, Nrf2, nucleolin, phospho (p)-ATM (Ser 1981 ), ATM, Chk1, Chk2, p-Chk2 (Thr 68 ), Cdc25c, p-Cdc25C (Ser 216 ), ubiquitin, procaspase-3, procaspase-9, Cdk2, Bad, Beclin-1, LC3, and Atg-7 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Western Blot, Control, Cell Culture, SDS Page, Phospho-proteomics, Transfection, Ubiquitin Proteomics, High Molecular Weight, Flow Cytometry

Journal: Oncotarget

Article Title: Camptothecin induces G 2 /M phase arrest through the ATM-Chk2-Cdc25C axis as a result of autophagy-induced cytoprotection: Implications of reactive oxygen species

doi: 10.18632/oncotarget.24934

Figure Lengend Snippet: ( A ) LNCaP cells were treated with 4 μM CPT for the indicated time points. Cell lysates were resolved on SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and probed with antibodies against p-ERK, ERK, p-p38, p38, p-JNK, and JNK. ( B ) The LNCaP cells were stimulated with CPT 4 μM for indicated time points after pretreatment with 20 μM SP600125 (SP), 20 μM PD98059 (PD), and 10 μM SB203580 (SB) for 1 h. Cell lysates were resolved on SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and probes with antibodies against Cdc25C, p-Cdc25C, cyclin B1, and p21. β-Actin was used as an internal control. ( C ) LNCaP cells were stimulated with 4 μM CPT for the indicated time after pretreatment with SP600125 (20 μM and 40 μM), PD98059 (10 μM and 20 μM), and SB203580 (5 μM and 10 μM) for 1 h. The cells were stained with PI and analyzed by flow cytometry at 12 h ( top ) and 24 h ( bottom ).

Article Snippet: Antibodies against Cdk2, cyclin B1, p21, β-actin, Nrf2, nucleolin, phospho (p)-ATM (Ser 1981 ), ATM, Chk1, Chk2, p-Chk2 (Thr 68 ), Cdc25c, p-Cdc25C (Ser 216 ), ubiquitin, procaspase-3, procaspase-9, Cdk2, Bad, Beclin-1, LC3, and Atg-7 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Control, Staining, Flow Cytometry