anti-pchk1 antibody #2348bf  (Cell Signaling Technology Inc)

Journal: iScience

Article Title: IMPDH inhibition induces DNA replication stress and ATR sensitivity in Merkel cell carcinoma

doi: 10.1016/j.isci.2025.112567

Figure Lengend Snippet: Activation of p53 by IMPDH inhibition is dependent on RS (A) Immunoblot of WaGa cells treated with MPA (1 μM) for indicated times. Representative of 3 independent experiments. (B) Immunoblot of WaGa cells treated with MPA (1 μM) and guanosine (10 μM) for 24 h. Representative of 3 independent experiments. (C) Immunoblot of WaGa cells treated for 24 h with MPA (1 μM) concurrent with inhibitors for ATR (250 nM; berzosertib), ATM (10 μM; KU55933), DNA-PK (10 μM; AZD7648) or with all three inhibitors combined (3x). Representative of 3 independent experiments. For panels A-C, the total KAP1 blot is a reblot of the pKAP1 blot, the total CHK1 blot is a reblot of pCHK1, and the total CHK2 blot is a reblot of pCHK2.

Article Snippet: pCHK1 (D7H2; Ser317) , Cell Signaling Technology , CAT# 8191; RRID: AB_10859365.

Techniques: Activation Assay, Inhibition, Western Blot

Journal: iScience

Article Title: IMPDH inhibition induces DNA replication stress and ATR sensitivity in Merkel cell carcinoma

doi: 10.1016/j.isci.2025.112567

Figure Lengend Snippet: Dual inhibition of IMPDH and ATR induces p53-independent replication catastrophe (A) Immunoblot of WaGa cells pre-induced with DOX (1 μg/mL) for 24 h to express p53DD or eGFP followed by treatment with MPA (1 μM) and berzosertib (250 nM) for an additional 24 h. Representative of 3 independent experiments. (B) Immunoblot of MKL-1 p53 KO or control (AAVS1) cells treated with MPA (1 μM) and berzosertib (250 nM) for 3 days. Representative of 3 independent experiments. For panels A and B, the total p53 blot is a reblot of pp53, the total KAP1 blot is a reblot of the pKAP1 blot, the total CHK1 blot is a reblot of pCHK1, and the total RPA32 blot is a reblot of pRPA32. (C) Flow cytometry analysis of chromatin-associated γH2AX and RPA32 in WaGa cells pre-induced and treated as in (A). Gating strategy for each defined population is shown. Images generated in FlowJo. Representative of 3 independent experiments. (D) Quantification of populations from (C). N = 3; mean ± SD. (E) Quantification of chromatin-associated CDC45 from WaGa cells treated as in (A). N = 3; mean ± SD; two-way ordinary ANOVA corrected for multiple comparisons via Tukey post hoc test; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (F) Quantification of chromatin-associated PCNA from WaGa cells treated as in (A). N = 3; mean ± SD; statistical tests are identical to (E). (G) Quantification of WaGa single cells dual-positive for AV and DAPI prepared as in (A) but treated with inhibitors for 2 days. N = 3; mean ± SD; statistical tests are identical to (E).

Article Snippet: pCHK1 (D7H2; Ser317) , Cell Signaling Technology , CAT# 8191; RRID: AB_10859365.

Techniques: Inhibition, Western Blot, Control, Flow Cytometry, Generated

Journal: NAR Cancer

Article Title: PARP inhibitor response is enhanced in prostate cancer when XRCC1 expression is reduced

doi: 10.1093/narcan/zcaf015

Figure Lengend Snippet: Antibodies used for immunoblotting experiments

Article Snippet: pCHK1-S345 (#2348) , 1:500 , Cell Signaling Technology.

Techniques: Western Blot

Journal: Biomolecules & Therapeutics

Article Title: Dual Disruption of DNA Repair by a Novel CHK2 Inhibitor, ART-446, and Olaparib is a Promising Strategy for Triple-Negative Breast Cancer Therapy

doi: 10.4062/biomolther.2025.029

Figure Lengend Snippet: Synergistic effects of ART-446 and olaparib in triple-negative breast cancer cell lines. (A) Synergistic effects of a CHK2 inhibitor (ART-446) and a PARP inhibitor (olaparib). Triple-negative breast cancer cells were coincubated with ART-446 and olaparib at the indicated concentrations for 72 h. (B) Western blot analysis of RAD51, γH2AX, pCHK2 (Ser516), pCHK2 (Thr68), pCHK1 (Ser296), cleaved PARP1 (after 96 h of treatment), and β-actin in MDA-MB-436 cells following 24 h of treatment with ART-446 and olaparib at the concentrations indicated at the top.

Article Snippet: After 1h blocked with 5% bovine serum albumin (Affimetrix, Santa Clara, CA, USA) at room temperature, the membranes were incubated with primary antibodies against BRCA1 (Cell Signaling, #9010, Danvers, MA, USA), RAD51 (Cell Signaling, #8875), γH2AX (Cell Signaling, #7631), pCHK2 (Thr68 or Ser516) (Cell Signaling, #2661 or #2669), pCHK1 (Ser296) (Cell Signaling, #2349) or pCDC2 (Tyr15) (Cell Signaling, #9111) at 4°C overnight.

Techniques: Western Blot

Journal: Biomolecules & Therapeutics

Article Title: Dual Disruption of DNA Repair by a Novel CHK2 Inhibitor, ART-446, and Olaparib is a Promising Strategy for Triple-Negative Breast Cancer Therapy

doi: 10.4062/biomolther.2025.029

Figure Lengend Snippet: Differential sensitivity of BRCA2 knockout cells to ART-446 and olaparib treatment. (A) Dose‒response curves for the viability of DLD-1 wild-type (WT) and BRCA2 knockout (KO) cells after treatment with ART-446 or olaparib. Compared with WT cells, BRCA2-KO cells were more sensitive to both treatments. (B) Western blot analysis showing the expression of BRCA1, RAD51, γH2AX, pCHK2 (Ser516, Thr68), pCHK1 (Ser296), pCDC2 (Tyr15), and β-actin in WT and BRCA2 KO cells following 24 h of treatment with ART-446 at the indicated concentrations.

Article Snippet: After 1h blocked with 5% bovine serum albumin (Affimetrix, Santa Clara, CA, USA) at room temperature, the membranes were incubated with primary antibodies against BRCA1 (Cell Signaling, #9010, Danvers, MA, USA), RAD51 (Cell Signaling, #8875), γH2AX (Cell Signaling, #7631), pCHK2 (Thr68 or Ser516) (Cell Signaling, #2661 or #2669), pCHK1 (Ser296) (Cell Signaling, #2349) or pCDC2 (Tyr15) (Cell Signaling, #9111) at 4°C overnight.

Techniques: Knock-Out, Western Blot, Expressing

Journal: Biomolecules & Therapeutics

Article Title: Dual Disruption of DNA Repair by a Novel CHK2 Inhibitor, ART-446, and Olaparib is a Promising Strategy for Triple-Negative Breast Cancer Therapy

doi: 10.4062/biomolther.2025.029

Figure Lengend Snippet: Antitumor activity of ART-446 in combination with olaparib in MDA-MB-436 xenografts. (A) Xenograft experiments were performed using the MDA-MB-436 cell line in athymic nude mice. Four experimental groups were studied: control (n=10), ART-446 alone (n=10), olaparib alone (n=10), and ART-446 combined with olaparib (n=10). Tumor volumes were measured twice per week. The body weights of the mice were monitored throughout the treatment period. The values are expressed as means ± standard deviations. The statistical significance of the difference in tumor volume between different groups at different times was analyzed via two-way ANOVA with the Holm‒Sidak multiple comparison test (** p <0.01; *** p <0.001; ns: nonsignificant). (B) Immunohistochemistry of MDA-MB-436 xenograft FFPE sections was used to assess pCHK1, pCHK2, pCDC2, Ki-67, pH3, γH2AX, cleaved caspase-3 and cleaved PARP expression. Percentage of immunoreactive cells is shown as the mean ± SD of eight tumor mass histological fields. ** p <0.01 compared with olaparib alone (Tukey’s honestly significant difference test).

Article Snippet: After 1h blocked with 5% bovine serum albumin (Affimetrix, Santa Clara, CA, USA) at room temperature, the membranes were incubated with primary antibodies against BRCA1 (Cell Signaling, #9010, Danvers, MA, USA), RAD51 (Cell Signaling, #8875), γH2AX (Cell Signaling, #7631), pCHK2 (Thr68 or Ser516) (Cell Signaling, #2661 or #2669), pCHK1 (Ser296) (Cell Signaling, #2349) or pCDC2 (Tyr15) (Cell Signaling, #9111) at 4°C overnight.

Techniques: Activity Assay, Control, Comparison, Immunohistochemistry, Expressing