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Cell Signaling Technology Inc anti phosphorylated chk2

Fig. 7. PCM1 promotes CHK1 activation to maintain cell survival upon prolonged replication stress. (A) Depletion of PCM1 did not affect <t>CHK2</t> activation upon HU treatment. Extracts of control or PCM1-depleted cells were analyzed by western blotting with antibodies against phosphorylated CHK2, CHK2, and tubulin (loading control). (B) Inhibition of CHK2 did not affect U2-OS cell viability upon HU treatment. Quantitative results of relative cell numbers of HU-treated U2-OS cells in the presence of CHK2 inhibitor (CHK2i). (C) Depletion of PCM1 reduced CHK1 activation upon HU treatment. Extracts of control or PCM1-depleted cells were analyzed by western blotting with antibodies against phosphorylated CHK1, CHK1, and tubulin (loading control). (B) Inhibition of CHK1 reduced U2-OS cell viability upon HU treatment. Quantitative results of relative cell numbers of HU-treated U2-OS cells in the presence of CHK1 inhibitor (CHK1i). n.s.: no significance, ***: P < 0.001. Each experiment was repeated at least three times.

Anti Phosphorylated Chk2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1368 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 96 stars, based on 1368 article reviews

anti phosphorylated chk2 - by Bioz Stars, 2026-09

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1) Product Images from "Pericentriolar material 1 aggregation maintains cell survival upon prolonged replication stress."

Article Title: Pericentriolar material 1 aggregation maintains cell survival upon prolonged replication stress.

Journal: Archives of biochemistry and biophysics

doi: 10.1016/j.abb.2025.110383

Fig. 7. PCM1 promotes CHK1 activation to maintain cell survival upon prolonged replication stress. (A) Depletion of PCM1 did not affect CHK2 activation upon HU treatment. Extracts of control or PCM1-depleted cells were analyzed by western blotting with antibodies against phosphorylated CHK2, CHK2, and tubulin (loading control). (B) Inhibition of CHK2 did not affect U2-OS cell viability upon HU treatment. Quantitative results of relative cell numbers of HU-treated U2-OS cells in the presence of CHK2 inhibitor (CHK2i). (C) Depletion of PCM1 reduced CHK1 activation upon HU treatment. Extracts of control or PCM1-depleted cells were analyzed by western blotting with antibodies against phosphorylated CHK1, CHK1, and tubulin (loading control). (B) Inhibition of CHK1 reduced U2-OS cell viability upon HU treatment. Quantitative results of relative cell numbers of HU-treated U2-OS cells in the presence of CHK1 inhibitor (CHK1i). n.s.: no significance, ***: P < 0.001. Each experiment was repeated at least three times.

Figure Legend Snippet: Fig. 7. PCM1 promotes CHK1 activation to maintain cell survival upon prolonged replication stress. (A) Depletion of PCM1 did not affect CHK2 activation upon HU treatment. Extracts of control or PCM1-depleted cells were analyzed by western blotting with antibodies against phosphorylated CHK2, CHK2, and tubulin (loading control). (B) Inhibition of CHK2 did not affect U2-OS cell viability upon HU treatment. Quantitative results of relative cell numbers of HU-treated U2-OS cells in the presence of CHK2 inhibitor (CHK2i). (C) Depletion of PCM1 reduced CHK1 activation upon HU treatment. Extracts of control or PCM1-depleted cells were analyzed by western blotting with antibodies against phosphorylated CHK1, CHK1, and tubulin (loading control). (B) Inhibition of CHK1 reduced U2-OS cell viability upon HU treatment. Quantitative results of relative cell numbers of HU-treated U2-OS cells in the presence of CHK1 inhibitor (CHK1i). n.s.: no significance, ***: P < 0.001. Each experiment was repeated at least three times.

Techniques Used: Activation Assay, Control, Western Blot, Inhibition

Ubiquitin Proteomics:

Article Title: ROS-ATM-CHK2 axis stabilizes HIF-1α and promotes tumor angiogenesis in hypoxic microenvironment
Article Snippet: Antibodies against CHK2 (05-649) were purchased from Merck KGaA (Darmstadt, Germany). .. Antibodies targeting phosphorylated CHK2 (Thr68, #2661), ATM (#2873), p-ATM (Ser1981, #13050), VEGF-A (#50661), ubiquitin (#3933S), β-actin (#3700), human influenza hemagglutinin (HA)-tag (#2367) and His-tag (#2365) were sourced from Cell Signaling Technology, Inc. (Danvers, MA, USA). ..

Article Title: ROS-ATM-CHK2 axis stabilizes HIF-1α and promotes tumor angiogenesis in hypoxic microenvironment.
Article Snippet: Antibodies against CHK2 (05-649) were purchased from Merck KGaA (Darmstadt, Germany). .. Antibodies targeting phosphorylated CHK2 (Thr68, #2661), ATM (#2873), p-ATM (Ser1981, #13050), VEGF-A (#50661), ubiquitin (#3933S), β-actin (#3700), human influenza hemagglutinin (HA)-tag (#2367) and His-tag (#2365) were sourced from Cell Signaling Technology, Inc. (Danvers, MA, USA). ..

Staining:

Article Title: Replication stress response biomarkers for immunotherapy response
Article Snippet: Antibodies against cyclin E (HE12; sc-247), p21 (C-19; sc-397), CHK1 (G4; sc-8408), and p53 (DO-1; sc-126) were purchased from Santa Cruz Biotechnology. .. Antibodies against CHK2 (2662), phosphorylated CHK2 (Thr68; 2661), phosphorylated p53 (Ser15; 9284), and a senescence β-galactosidase staining kit (9860) were purchased from Cell Signaling Technology. .. Antibodies against cyclin E (HE12; sc-247), p21 (C-19; sc-397), CHK1 (G4; sc-8408), and p53 (DO-1; sc-126) were purchased from Santa Cruz Biotechnology.

Membrane:

Article Title: MYH knockdown in pancreatic cancer cells creates an exploitable DNA repair vulnerability
Article Snippet: .. The membrane was blocked with 5 % skim milk powder in PBS/0.1 % Tween-20 (PBST) for 1 h at room temperature and probed with antibodies against the following targets: i) MYH (1:1000) mouse monoclonal antibody (Abnova, Cat. H00004595-M01). ii) Phosphorylated Chk1 (1:1000) rabbit monoclonal antibody (Cell Signaling, Cat.2348). iii) Phosphorylated Chk2 (1:500) rabbit polyclonal antibody (Cell Signaling, Cat2661). iv) MTH1 (1:1000) monoclonal mouse antibody (Santa Cruz Biotechnology, Cat. sc-271082). v) OGG1 (1:1000) rabbit polyclonal antibody (Novus Biologicals, Cat. NB100-106). vi) MSH2 (1:1000) rabbit polyclonal antibody (Abcam, Cat. 70270). vii) GAPDH (1:50000) monoclonal mouse antibody (Abcam, Cat. Ab8245). viii) α-tubulin (1:1000) monoclonal mouse antibody (Sigma-Aldrich, Cat. T9026). .. Primary antibodies were detected with horseradish peroxidase (HRP)-linked polyclonal goat anti-mouse IgG (DAKO, Cat. PO447) or HRP-linked polyclonal goat anti-rabbit IgG (DAKO, Cat. PO448) diluted in 5 % skim milk/PBST, for 1 h at room temperature.

Article Title: MYH knockdown in pancreatic cancer cells creates an exploitable DNA repair vulnerability.
Article Snippet: Pancreatic ductal adenocarcinoma (PDAC) has a poor 5-year survival rate of just 13 %.. Conventional therapies fail due to acquired chemoresistance.. We previously identified MutY-Homolog (MYH), a protein that repairs oxidative DNA damage, as a therapeutic target that induces apoptosis in PDAC cells.

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Journal: Archives of biochemistry and biophysics

Article Title: Pericentriolar material 1 aggregation maintains cell survival upon prolonged replication stress.

doi: 10.1016/j.abb.2025.110383

Figure Lengend Snippet: Fig. 7. PCM1 promotes CHK1 activation to maintain cell survival upon prolonged replication stress. (A) Depletion of PCM1 did not affect CHK2 activation upon HU treatment. Extracts of control or PCM1-depleted cells were analyzed by western blotting with antibodies against phosphorylated CHK2, CHK2, and tubulin (loading control). (B) Inhibition of CHK2 did not affect U2-OS cell viability upon HU treatment. Quantitative results of relative cell numbers of HU-treated U2-OS cells in the presence of CHK2 inhibitor (CHK2i). (C) Depletion of PCM1 reduced CHK1 activation upon HU treatment. Extracts of control or PCM1-depleted cells were analyzed by western blotting with antibodies against phosphorylated CHK1, CHK1, and tubulin (loading control). (B) Inhibition of CHK1 reduced U2-OS cell viability upon HU treatment. Quantitative results of relative cell numbers of HU-treated U2-OS cells in the presence of CHK1 inhibitor (CHK1i). n.s.: no significance, ***: P < 0.001. Each experiment was repeated at least three times.

Article Snippet: The following antibodies were used for immunofluorescence and Western blot assays: anti-γ-tubulin (Sigma), anti-PCM1, anti-phosphorylated ATR, anti-ATR, anti-CHK1, anti-CHK2, anti-phosphorylated CHK1, anti-phosphorylated CHK2, anti-LC3 A/B (Cell Signaling), antip150glued, anti-GM130 (BD Biosciences), anti-Ku70, anti-α-tubulin, antiATM, anti-β-actin (GeneTex), anti-γ-H2AX, anti-p-ATM (Abcam), antiphosphorylated DNA-PKcs, anti-DNA-PKcs (Santa Cruz Biotech).

Techniques: Activation Assay, Control, Western Blot, Inhibition

A Western blotting of HIF-1α, p-CHK2 Thr68, CHK2, VEGF. For all Western blotting, β-actin was used as a loading control. 1% O 2 was performed in HEK293 cells and HEK293-CHK2-KO cells for indicated times. B Western blotting of HIF-1α, p-CHK2 Thr68 and CHK2. HEK293 cells were stimulated by 1% O 2 , CHK2 inhibitor and combination for 6 h. C , D Flow cytometric analysis of ROS levels and median fluorescence intensity of ROS expression. H1299 cells were stimulated by 1% O 2 with or without NAC for 6 h. ** p < 0.01. E Western blotting of p-ATM S1981, ATM, HIF-1α, p-CHK2 Thr68 and CHK2. HEK293 cells were pretreated with NAC for 4 h and then cultured in 1% O 2 for 6 h. F Western blotting of p-ATM S1981, ATM, HIF-1α, p-CHK2 Thr68 and CHK2. H1299 cells were transfected with the indicated shRNA and exposed to 1% O 2 for indicated times. G Western blotting of p-ATM S1981, ATM, HIF-1α, p-CHK2 Thr68 and CHK2. HEK293 and HEK293-CHK2-KO cells were exposed to 1% O 2 or combination with ATM inhibitor for 6 h. RT-qPCR analysis of PKM2 ( H ), PDK1 ( I ) and BNIP3 ( J ) mRNA in CHK2-deficient or wildtype HEK293 cells treated with or without hypoxia for 6 h. ** p < 0.01, n.s. not significant. K Dual-luciferase reporter gene experiment of PKM2, PDK1 and BNIP3. HEK293-CHK2-KO cells were co-transfected with reporter gene and CHK2 plasmid under normoxia (21% O 2 ) and hypoxia (1% O 2 ) . ** p < 0.01, n.s. not significant.

Journal: Oncogene

Article Title: ROS-ATM-CHK2 axis stabilizes HIF-1α and promotes tumor angiogenesis in hypoxic microenvironment

doi: 10.1038/s41388-025-03336-w

Figure Lengend Snippet: A Western blotting of HIF-1α, p-CHK2 Thr68, CHK2, VEGF. For all Western blotting, β-actin was used as a loading control. 1% O 2 was performed in HEK293 cells and HEK293-CHK2-KO cells for indicated times. B Western blotting of HIF-1α, p-CHK2 Thr68 and CHK2. HEK293 cells were stimulated by 1% O 2 , CHK2 inhibitor and combination for 6 h. C , D Flow cytometric analysis of ROS levels and median fluorescence intensity of ROS expression. H1299 cells were stimulated by 1% O 2 with or without NAC for 6 h. ** p < 0.01. E Western blotting of p-ATM S1981, ATM, HIF-1α, p-CHK2 Thr68 and CHK2. HEK293 cells were pretreated with NAC for 4 h and then cultured in 1% O 2 for 6 h. F Western blotting of p-ATM S1981, ATM, HIF-1α, p-CHK2 Thr68 and CHK2. H1299 cells were transfected with the indicated shRNA and exposed to 1% O 2 for indicated times. G Western blotting of p-ATM S1981, ATM, HIF-1α, p-CHK2 Thr68 and CHK2. HEK293 and HEK293-CHK2-KO cells were exposed to 1% O 2 or combination with ATM inhibitor for 6 h. RT-qPCR analysis of PKM2 ( H ), PDK1 ( I ) and BNIP3 ( J ) mRNA in CHK2-deficient or wildtype HEK293 cells treated with or without hypoxia for 6 h. ** p < 0.01, n.s. not significant. K Dual-luciferase reporter gene experiment of PKM2, PDK1 and BNIP3. HEK293-CHK2-KO cells were co-transfected with reporter gene and CHK2 plasmid under normoxia (21% O 2 ) and hypoxia (1% O 2 ) . ** p < 0.01, n.s. not significant.

Article Snippet: Antibodies targeting phosphorylated CHK2 (Thr68, #2661), ATM (#2873), p-ATM (Ser1981, #13050), VEGF-A (#50661), ubiquitin (#3933S), β-actin (#3700), human influenza hemagglutinin (HA)-tag (#2367) and His-tag (#2365) were sourced from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Western Blot, Control, Fluorescence, Expressing, Cell Culture, Transfection, shRNA, Quantitative RT-PCR, Luciferase, Plasmid Preparation

A Western blotting of exogenous HA-HIF-1α and Flag-tagged CHK2. HA-tagged HIF-1α (3 μg) and Flag-tagged CHK2 (1 μg, 3 μg) was transiently transfected into H1299 cells and then exposed to 1% O 2 . B Western blotting of exogenous HA-HIF-1α and CHK2. HA-tagged HIF-1α was transiently transfected into HEK293 and HEK293-CHK2-KO cells under hypoxic microenvironment. MG132 and CQ was then added to above cells respectively or together as indicated. C Endogenous HIF-1α ubiquitination in HEK293 and HEK293-CHK2-KO cells under hypoxia (1% O 2 ) for 6 h. Exogenous HA-HIF-1α ubiquitination under normoxia ( D ) and hypoxia ( E ) in HEK293-CHK2-KO cells transiently transfected with indicated plasmids. F Western blotting of exogenous double mutant HIF-1α protein at both hydroxylation sites P402A/P564A (HA-HIF-1α-DM). HEK293 cells were transfected with an increasing amount of Flag-CHK2 expression plasmid and exposed to hypoxia. G Western blotting of exogenous triple mutant HIF-1α protein at P402A/P564A/N803A (HA-HIF-1α-TM). HEK293 cells were transfected with an increasing amount of Flag-CHK2 expression plasmid and exposed to hypoxia.

Journal: Oncogene

Article Title: ROS-ATM-CHK2 axis stabilizes HIF-1α and promotes tumor angiogenesis in hypoxic microenvironment

doi: 10.1038/s41388-025-03336-w

Figure Lengend Snippet: A Western blotting of exogenous HA-HIF-1α and Flag-tagged CHK2. HA-tagged HIF-1α (3 μg) and Flag-tagged CHK2 (1 μg, 3 μg) was transiently transfected into H1299 cells and then exposed to 1% O 2 . B Western blotting of exogenous HA-HIF-1α and CHK2. HA-tagged HIF-1α was transiently transfected into HEK293 and HEK293-CHK2-KO cells under hypoxic microenvironment. MG132 and CQ was then added to above cells respectively or together as indicated. C Endogenous HIF-1α ubiquitination in HEK293 and HEK293-CHK2-KO cells under hypoxia (1% O 2 ) for 6 h. Exogenous HA-HIF-1α ubiquitination under normoxia ( D ) and hypoxia ( E ) in HEK293-CHK2-KO cells transiently transfected with indicated plasmids. F Western blotting of exogenous double mutant HIF-1α protein at both hydroxylation sites P402A/P564A (HA-HIF-1α-DM). HEK293 cells were transfected with an increasing amount of Flag-CHK2 expression plasmid and exposed to hypoxia. G Western blotting of exogenous triple mutant HIF-1α protein at P402A/P564A/N803A (HA-HIF-1α-TM). HEK293 cells were transfected with an increasing amount of Flag-CHK2 expression plasmid and exposed to hypoxia.

Techniques: Western Blot, Transfection, Ubiquitin Proteomics, Mutagenesis, Expressing, Plasmid Preparation

A Colocalization of CHK2 and HIF-1α shown by representative confocal microscopic images of NSCLC tissues and H1299 cells. For H1299 cells, experiments were conducted under normoxia (21% O 2 ) and hypoxia (1% O 2 ), respectively. B , C . Co-IP of endogenous CHK2 with HIF-1α in H1299 cells under normoxia (21% O 2 ) and hypoxia (1% O 2 ). IgG, immunoglobulin G. D Co-IP of endogenous CHK2 with HIF-1α in H1299 cells pretreated with NAC for 4 h and then cultured in 1% O 2 for 6 h. E GST-pull-down assay of His-HIF-1α fusion proteins with bacterially expressed GST-CHK2 fragments. Coomassie Brilliant Blue (CBB) staining shows expression of GST and GST-CHK2 fragments. F GST-pull-down assay of His-CHK2 fusion proteins with bacterially expressed GST-HIF-1α fragments. CBB staining shows expression of GST and GST-HIF-1α fragments. G , H Co-IP of exogenous HA-HIF-1α with Flag-CHK2 under normoxia (21% O 2 ) and hypoxia (1% O 2 ) in HEK293-CHK2-KO cells transiently transfected with indicated plasmids.

Journal: Oncogene

Article Title: ROS-ATM-CHK2 axis stabilizes HIF-1α and promotes tumor angiogenesis in hypoxic microenvironment

doi: 10.1038/s41388-025-03336-w

Figure Lengend Snippet: A Colocalization of CHK2 and HIF-1α shown by representative confocal microscopic images of NSCLC tissues and H1299 cells. For H1299 cells, experiments were conducted under normoxia (21% O 2 ) and hypoxia (1% O 2 ), respectively. B , C . Co-IP of endogenous CHK2 with HIF-1α in H1299 cells under normoxia (21% O 2 ) and hypoxia (1% O 2 ). IgG, immunoglobulin G. D Co-IP of endogenous CHK2 with HIF-1α in H1299 cells pretreated with NAC for 4 h and then cultured in 1% O 2 for 6 h. E GST-pull-down assay of His-HIF-1α fusion proteins with bacterially expressed GST-CHK2 fragments. Coomassie Brilliant Blue (CBB) staining shows expression of GST and GST-CHK2 fragments. F GST-pull-down assay of His-CHK2 fusion proteins with bacterially expressed GST-HIF-1α fragments. CBB staining shows expression of GST and GST-HIF-1α fragments. G , H Co-IP of exogenous HA-HIF-1α with Flag-CHK2 under normoxia (21% O 2 ) and hypoxia (1% O 2 ) in HEK293-CHK2-KO cells transiently transfected with indicated plasmids.

Techniques: Co-Immunoprecipitation Assay, Cell Culture, Pull Down Assay, Staining, Expressing, Transfection

A Mass spectrogram of phosphorylation sites of HIF-1α protein T645. B Sequence alignment surrounding the T645 residue of HIF-1α homologs in various species. C Western blotting of HA-HIF-1α, p-CHK2 and CHK2. CHK2 inhibitor was added to HEK2923 cells transiently transfected with plasmids as indicated under hypoxia (1% O 2 ). D Western blotting of HA-HIF-1α and CHK2. shCHK2, HA-HIF-1α and HA-HIF-1α Thr645A plasmids was transiently transfected into HEK293 cells as indicated under hypoxia (1% O 2 ). E Representative images of HUVEC tube formation assay after 24 h incubation in the indicated conditioned media from H1299 cells. F Mean tube length in E was quantified. ** p < 0.01, n.s. not significant. G Exogenous HA-HIF-1α ubiquitination was detected by Co-IP. Flag-CHK2, HA-HIF-1α and HA-HIF-1α Thr645A plasmids were transiently transfected into HEK293 cells as indicated under hypoxia (1% O 2 ). H Co-IP of endogenous HIF-1α with USP7 in HEK293 cells and HEK293-CHK2-KO cells under normoxia (21% O 2 ) and hypoxia (1% O 2 ). I Co-IP of endogenous HIF-1α with USP7. Flag-CHK2 plasmids was transiently transfected into HEK293 cells as indicated under normoxia (21% O 2 ) and hypoxia (1% O 2 ). J Ubiquitination of Flag-HIF-1α WT and Flag-HIF-1α T645A. Flag-HIF-1α and Flag-HIF-1α Thr645A plasmids were transiently transfected into HEK293 cells as indicated under hypoxia (1% O 2 ).

Journal: Oncogene

Article Title: ROS-ATM-CHK2 axis stabilizes HIF-1α and promotes tumor angiogenesis in hypoxic microenvironment

doi: 10.1038/s41388-025-03336-w

Figure Lengend Snippet: A Mass spectrogram of phosphorylation sites of HIF-1α protein T645. B Sequence alignment surrounding the T645 residue of HIF-1α homologs in various species. C Western blotting of HA-HIF-1α, p-CHK2 and CHK2. CHK2 inhibitor was added to HEK2923 cells transiently transfected with plasmids as indicated under hypoxia (1% O 2 ). D Western blotting of HA-HIF-1α and CHK2. shCHK2, HA-HIF-1α and HA-HIF-1α Thr645A plasmids was transiently transfected into HEK293 cells as indicated under hypoxia (1% O 2 ). E Representative images of HUVEC tube formation assay after 24 h incubation in the indicated conditioned media from H1299 cells. F Mean tube length in E was quantified. ** p < 0.01, n.s. not significant. G Exogenous HA-HIF-1α ubiquitination was detected by Co-IP. Flag-CHK2, HA-HIF-1α and HA-HIF-1α Thr645A plasmids were transiently transfected into HEK293 cells as indicated under hypoxia (1% O 2 ). H Co-IP of endogenous HIF-1α with USP7 in HEK293 cells and HEK293-CHK2-KO cells under normoxia (21% O 2 ) and hypoxia (1% O 2 ). I Co-IP of endogenous HIF-1α with USP7. Flag-CHK2 plasmids was transiently transfected into HEK293 cells as indicated under normoxia (21% O 2 ) and hypoxia (1% O 2 ). J Ubiquitination of Flag-HIF-1α WT and Flag-HIF-1α T645A. Flag-HIF-1α and Flag-HIF-1α Thr645A plasmids were transiently transfected into HEK293 cells as indicated under hypoxia (1% O 2 ).

Techniques: Phospho-proteomics, Sequencing, Residue, Western Blot, Transfection, HUVEC Tube Formation Assay, Incubation, Ubiquitin Proteomics, Co-Immunoprecipitation Assay

A Ten mice (n = 5/group) were injected with H1299 cells. DMSO or the CHK2 inhibitor (1 mg/kg) was administered intraperitoneally on alternate days for 20 days. B Representative CD31 and Ki-67 staining of harvested xenografts of mice treated by DMSO or CHK2 inhibitor. Scale bar = 50 μm. C Quantification of IHC. ** p < 0.01. D Western blotting of HA-tagged protein in HIF-1α-WT and HIF-1α-T645A stable transduced H1299 cells. E , F Flow cytometry and statistical analysis showing cell apoptosis exposed to hypoxia of the H1299 cells with indicated transfection. ** p < 0.01. G H1299 cells stably expressing HIF-1α WT or HIF-1α T645A were injected subcutaneously into the rear flanks of athymic nude mice ( n = 6). H IHC of harvested tumors in G with antibodies specific for CD31 and Ki-67 to assess angiogenesis and proliferation respectively. I Measurement of tumor volume of mice in ( G ). ** p < 0.01. J Representative CHK2 and CD31 staining in NSCLC tissues, n = 50. K Scattering plot depicting the correlation between CHK2 and CD31 score.

Journal: Oncogene

Article Title: ROS-ATM-CHK2 axis stabilizes HIF-1α and promotes tumor angiogenesis in hypoxic microenvironment

doi: 10.1038/s41388-025-03336-w

Figure Lengend Snippet: A Ten mice (n = 5/group) were injected with H1299 cells. DMSO or the CHK2 inhibitor (1 mg/kg) was administered intraperitoneally on alternate days for 20 days. B Representative CD31 and Ki-67 staining of harvested xenografts of mice treated by DMSO or CHK2 inhibitor. Scale bar = 50 μm. C Quantification of IHC. ** p < 0.01. D Western blotting of HA-tagged protein in HIF-1α-WT and HIF-1α-T645A stable transduced H1299 cells. E , F Flow cytometry and statistical analysis showing cell apoptosis exposed to hypoxia of the H1299 cells with indicated transfection. ** p < 0.01. G H1299 cells stably expressing HIF-1α WT or HIF-1α T645A were injected subcutaneously into the rear flanks of athymic nude mice ( n = 6). H IHC of harvested tumors in G with antibodies specific for CD31 and Ki-67 to assess angiogenesis and proliferation respectively. I Measurement of tumor volume of mice in ( G ). ** p < 0.01. J Representative CHK2 and CD31 staining in NSCLC tissues, n = 50. K Scattering plot depicting the correlation between CHK2 and CD31 score.

Techniques: Injection, Staining, Paraffin-embedded Immunohistochemistry, Western Blot, Flow Cytometry, Transfection, Stable Transfection, Expressing

Model showing CHK2 interaction with the HIF-1α inhibitory domain and phosphorylate HIF-1α at Thr645 site, enhancing HIF-1α binding to its deubiquitinating enzyme USP7, prohibiting HIF-1α ubiquitination and thus triggering angiogenesis by upregulating VEGF.

Journal: Oncogene

Article Title: ROS-ATM-CHK2 axis stabilizes HIF-1α and promotes tumor angiogenesis in hypoxic microenvironment

doi: 10.1038/s41388-025-03336-w

Figure Lengend Snippet: Model showing CHK2 interaction with the HIF-1α inhibitory domain and phosphorylate HIF-1α at Thr645 site, enhancing HIF-1α binding to its deubiquitinating enzyme USP7, prohibiting HIF-1α ubiquitination and thus triggering angiogenesis by upregulating VEGF.

Techniques: Binding Assay, Ubiquitin Proteomics

A In vitro kinase assays were performed with different concentrations of His-tagged human APE1 (His-hAPE1) as indicated, Flag-tagged WT human ATM (Flag-hATM) and His-tagged kinase-deficient human Chk2 (His-hChk2), with DMSO or ATM inhibitor KU55933 (1 mM). ATM kinase activity was indicated by Chk2 phosphorylation (P-hChk2) via immunoblotting using anti-Chk2 T68 phosphorylation specific antibodies. B Kinase assays were performed with different concentrations of His-hAPE1 as indicated, WT or KD (kinase-dead) Flag-hATM and His-hChk2. ATM kinase activity was indicated by P-hChk2. C Kinase assays were performed with Control (PBS), GST, WT or ΔNT34 GST- Xenopus APE1, Flag-hATM and His-hChk2, with the addition of DMSO or KU55933 (1 mM). ATM kinase activity was indicated by P-hChk2. D Kinase assays were performed with Control (PBS), GST, GST- Xenopus APE1 WT or ΔNT34, Flag-tagged WT or kinase-dead (KD) ATM and His-hChk2. ATM kinase activity was indicated by P-hChk2. E Quantification and statistical analysis of the ratio of P-hChk2 vs. hChk2 from Panel C . Data are presented as mean values ± SD. ** p (GST vs GST-APE1-WT) = 0.0001; ** p (GST-APE1-WT vs GST-APE1-ΔNT34) = 0.0016; two-tailed, unpaired t-test, n = 3. F Kinase assays were performed with different concentrations of His-tagged APE1 NT34 (His-APE1-NT34) and DMSO/KU55933 as indicated. Chk2 phosphorylation was examined via immunoblotting analysis. The data presented in Panel A – D and F are representative of three biological replicates. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Distinct regulation of ATM signaling by DNA single-strand breaks and APE1

doi: 10.1038/s41467-024-50836-6

Figure Lengend Snippet: A In vitro kinase assays were performed with different concentrations of His-tagged human APE1 (His-hAPE1) as indicated, Flag-tagged WT human ATM (Flag-hATM) and His-tagged kinase-deficient human Chk2 (His-hChk2), with DMSO or ATM inhibitor KU55933 (1 mM). ATM kinase activity was indicated by Chk2 phosphorylation (P-hChk2) via immunoblotting using anti-Chk2 T68 phosphorylation specific antibodies. B Kinase assays were performed with different concentrations of His-hAPE1 as indicated, WT or KD (kinase-dead) Flag-hATM and His-hChk2. ATM kinase activity was indicated by P-hChk2. C Kinase assays were performed with Control (PBS), GST, WT or ΔNT34 GST- Xenopus APE1, Flag-hATM and His-hChk2, with the addition of DMSO or KU55933 (1 mM). ATM kinase activity was indicated by P-hChk2. D Kinase assays were performed with Control (PBS), GST, GST- Xenopus APE1 WT or ΔNT34, Flag-tagged WT or kinase-dead (KD) ATM and His-hChk2. ATM kinase activity was indicated by P-hChk2. E Quantification and statistical analysis of the ratio of P-hChk2 vs. hChk2 from Panel C . Data are presented as mean values ± SD. ** p (GST vs GST-APE1-WT) = 0.0001; ** p (GST-APE1-WT vs GST-APE1-ΔNT34) = 0.0016; two-tailed, unpaired t-test, n = 3. F Kinase assays were performed with different concentrations of His-tagged APE1 NT34 (His-APE1-NT34) and DMSO/KU55933 as indicated. Chk2 phosphorylation was examined via immunoblotting analysis. The data presented in Panel A – D and F are representative of three biological replicates. Source data are provided as a Source Data file.

Article Snippet: Antibodies against ATM phosphorylation at Ser1981 (Rockland Immunochemicals, Cat#200-301-500), Chk1 (Santa Cruz Biotechnology, Cat#sc-8408), Chk1 phosphorylation at Ser345 (Cell Signaling Technology, Cat#2348), Chk2 (Santa Cruz Biotechnology, Cat#sc-9064), Chk2 phosphorylation at Thr68 (Santa Cruz Biotechnology, Cat#sc-16297-R), Flag (Thermo Fisher, Cat#MA1-91878), GST (Santa Cruz Biotechnology, Cat#sc-138), H2AX (Cell Signaling Technology, Cat#7631 S), H2AX phosphorylation at Ser139 (Cell Signaling Technology, Cat#2577 S), H3 (Abcam, Cat#ab1791), His (Santa Cruz Biotechnology, Cat#sc-8036), Myc (Santa Cruz Biotechnology, Cat#sc-40), RPA32 phosphorylation at Ser33 (Bethyl Laboratories, Cat#A300-246A), and PCNA (Santa Cruz Biotechnology, Cat#sc-56) were purchased from commercially available vendors.

Techniques: In Vitro, Activity Assay, Western Blot, Control, Two Tailed Test

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anti phosphorylated chk2 (Cell Signaling Technology Inc)

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