mouse monoclonal anti parp  (Proteintech)

Journal: Journal of Advanced Research

Article Title: Circular RNA circATM binds PARP1 to suppress Wnt/β-catenin signaling and induce cell cycle arrest in gastric cancer cells

doi: 10.1016/j.jare.2025.04.033

Figure Lengend Snippet: Identification of PARP1 as a binding partner of circATM, and CircATM binds to the zinc-finger motif of the Ⅱ–Ⅲ domain of PARP1. (A, B, C) FLAG-tagged pLC5-circATM plasmids were successfully transfected into HEK293T cells (A). No protein expression was detected (B), while circATM expression increased > 1,000-fold post-transfection (C). (D) CircRNA pull-down assay was conducted using circATM and scramble probes, followed by silver staining. (E) Proteins pulled down by the circATM probes were identified by mass spectrometry; of these, 227 proteins were selected, based on having an iBAQ circATM probe to scramble probe ration of > 5.0. (F) Gene ontology analysis of the 227 selected proteins. (G) Protein-protein interaction network and MCODE components identified in the gene list corresponding to the 227 proteins. (H) PARP1 was identified as a binding partner of circATM by western blotting after protein mass spectrometry analysis. (I) The expression of PARP1 in TCGA STAD samples. (J) The expression of PARP1 in Clinical Proteomic Tumor Analysis Consortium (CPTAC) gastric cancer samples. (K) Kaplan-Meier curve of PARP1 gene expression in TCGA/GTEx STAD samples. (L) Detection of circATM in HEK293T cells transfected with vector pLC5-ciR by RT-qPCR and agarose gel electrophoresis after immunoprecipitation with IgG control and the anti-PARP1 antibody. (M) Detection of circATM in HEK293T cells transfected with the overexpression plasmid pLC5-circATM by RT-qPCR and agarose gel electrophoresis after immunoprecipitation with IgG control and the anti-PARP1 antibody. (N) Prediction of protein binding sites in the circATM nucleotide sequence. (O) Prediction of RNA binding sites in PARP1 amino acid sequence. (P) Construction of four PARP1 truncated constructs, including full-length PARP1 (aa 1–1,014), the PARP1 DNA-binding domain ZFⅠ–Ⅲ only (aa 1–360), PARP1 with a deleted DNA-binding domain ZFⅠ–Ⅲ (ΔZFⅠ–Ⅲ, aa 358–1,014), PARP1 with a deleted catalytic domain (Δsignature, aa 1–649). (Q) Detection of circATM/PARP1 complexes in HEK293T cells transfected with each of the four PARP1constructs at 48 h post-transfection by RT-qPCR after Co-IP. (R) Generation of PARP1 zinc-finger domain constructs, including full-length PARP1 (aa 1–1,014), ΔZFⅠ (aa 94–1,014), ΔZFⅠ–Ⅱ (aa 204–1,014), and ΔZFⅠ–Ⅲ (aa 358–1,014). (S) Detection of circATM binding to the different PARP1 zinc-finger domains in HEK293T cells transfected with each of the four constructs at 48 h post-transfection by RT-qPCR after Co-IP. * p ＜0.05, ** p ＜0.01, *** p ＜0.001.

Article Snippet: An anti-PARP1 monoclonal antibody from Proteintech Group (Cat# 66520–1-Ig, RRID:AB_2881883) was used in the RIP assay.

Techniques: Binding Assay, Transfection, Expressing, Pull Down Assay, Silver Staining, Mass Spectrometry, Western Blot, Gene Expression, Plasmid Preparation, Quantitative RT-PCR, Agarose Gel Electrophoresis, Immunoprecipitation, Control, Over Expression, Protein Binding, Sequencing, RNA Binding Assay, Construct, Co-Immunoprecipitation Assay

Journal: Journal of Advanced Research

Article Title: Circular RNA circATM binds PARP1 to suppress Wnt/β-catenin signaling and induce cell cycle arrest in gastric cancer cells

doi: 10.1016/j.jare.2025.04.033

Figure Lengend Snippet: CircATM binding to PARP1 inhibits Wnt/β-catenin signaling. (A) Western blotting was used to detect β-catenin levels after PARP1 immunoprecipitation in HEK293T cells transfected with vector pLC5-ciR and pLC5-circATM plasmids. (B) Western blotting analysis of circATM binding to β-catenin in the circRNA pull-down assay. (C) Western blotting analysis of PARP1 levels in HEK293T cells transfected with vector pLC5-ciR and pLC5-circATM plasmids after β-catenin immunoprecipitation. (D) TOPflash and FOPflash reporter assays were carried out to measure the transcriptional activity of TCF/β-catenin in AGS cells. (E) Wnt/β-catenin targets were detected by RT-qPCR after circATM siRNA transfection of AGS cells. (F) Wnt/β-catenin targets were detected by western blotting after circATM siRNA transfection of AGS cells. (G) The effect of circATM overexpression on Wnt/β-catenin targets in AGS cells transfected with si_circATM. * p ＜0.05, ** p ＜0.01, *** p ＜0.001.

Article Snippet: An anti-PARP1 monoclonal antibody from Proteintech Group (Cat# 66520–1-Ig, RRID:AB_2881883) was used in the RIP assay.

Techniques: Binding Assay, Western Blot, Immunoprecipitation, Transfection, Plasmid Preparation, Pull Down Assay, Activity Assay, Quantitative RT-PCR, Over Expression

Journal: Journal of Advanced Research

Article Title: Circular RNA circATM binds PARP1 to suppress Wnt/β-catenin signaling and induce cell cycle arrest in gastric cancer cells

doi: 10.1016/j.jare.2025.04.033

Figure Lengend Snippet: CircATM binding to PARP1 hinders DNA damage repair and induces cell cycle arrest. (A) Western blotting analysis of whether circATM directly bound to ATM in the circRNA pull-down assay. (B) Western blotting analysis of ATM levels after PARP1 immunoprecipitation in AGS and MKN45 cells transfected with negative control (NC) or circATM siRNA. (C) DNA damage response proteins were detected by western blot in AGS and MKN45 cells at 48 h after circATM siRNA transfection. (D) Analysis of cell-cycle-related proteins at 48 h after circATM siRNA transfection of AGS and MKN45 cells. (E) The effect of circATM overexpression on cell cycle proteins was detected by western blotting in AGS cells transfected with si_circATM. (F) Western blotting analysis of the effects of knockdown and overexpression of circATM on the half-life of PARP1 in AGS cells treated with 100 μg/ml CHX at different time points. (G) Western blotting analysis of the effects of knockdown and overexpression of circATM on PARP1 proteasomal degradation in AGS cells treated with 1 mM MG132 for 24 h.

Article Snippet: An anti-PARP1 monoclonal antibody from Proteintech Group (Cat# 66520–1-Ig, RRID:AB_2881883) was used in the RIP assay.

Techniques: Binding Assay, Western Blot, Pull Down Assay, Immunoprecipitation, Transfection, Negative Control, Over Expression, Knockdown

Journal: The Journal of Biological Chemistry

Article Title: Tubulin hyperacetylation drives HMGB1 nuclear exit via the ROS-PARP1 axis, leading to rotenone-induced G2/M arrest

doi: 10.1016/j.jbc.2025.110695

Figure Lengend Snippet: Rotenone triggers time-dependent HMGB1 nuclear exit coupled with increased PARylation. A , representative confocal images of time-dependent HMGB1 ( green ) localization in SH-SY5Y cells treated with rotenone (5μM) or DMSO control. DAPI ( blue ) indicates nuclear staining (scale bar = 10 μm). B , quantification of SH-SY5Y cells with cytoplasmic enrichment of HMGB1 shown in ( A ). Nucleus was defined by area of DAPI. Cytosolic HMGB1 was calculated by subtracting nuclear region of interest (ROI) from the area of green channel. n = 100 cells for each quantification. C , immunoblot analysis of HMGB1 protein levels in the nuclear and cytosolic fraction of SH-SY5Y cells treated with rotenone for 2, 4, 6 and 24 h and compared with DMSO treated controls. GAPDH was used as cytosolic loading control and lamin A/C was used as a nuclear loading control ( left panel ). The nuclear to cytoplasmic ratio was quantified using the lower band obtained in the cytoplasmic fraction of the gels ( right panel ). D , co-immunoprecipitation analysis was performed to assess the PARylation status of HMGB1 in whole-cell extracts following 24 h of rotenone treatment. Cells were immunoprecipitated with an anti-HMGB1 antibody, and PAR ( upper panel ) and HMGB1 ( middle panel ; low and high exposures) levels were analyzed. Additionally, cells were immunoprecipitated with an anti-PAR antibody, and HMGB1 levels ( lower panel ) were examined. E , immunoblot analysis of HMGB1 protein levels in the nuclear and cytosolic fraction of cells treated with rotenone for 24 h in the presence or absence of the PARP inhibitor PJ34 (50μM). GAPDH was used as cytosolic loading control and lamin A/C was used as a nuclear loading control ( left panel ). The nuclear to cytoplasmic ratio was quantified using the lower band obtained in the cytoplasmic fraction of the gels ( right panel ). F , representative confocal images of HMGB1 ( green ) and DAPI (blue) in SH-SY5Y cells treated with rotenone or DMSO control in the presence of PJ34. DAPI ( blue ) indicates nuclear staining (scale bar = 10 μm). G , quantification of cells with cytoplasmic enrichment of HMGB1. n = 100 cells for each quantification. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test (∗∗∗ p < 0.001; n = 3 biological replicates).

Article Snippet: Rabbit monoclonal antibody against PARP1 (#9532) was obtained from Cell Signaling Technology Inc. (CST).

Techniques: Control, Staining, Western Blot, Immunoprecipitation, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Tubulin hyperacetylation drives HMGB1 nuclear exit via the ROS-PARP1 axis, leading to rotenone-induced G2/M arrest

doi: 10.1016/j.jbc.2025.110695

Figure Lengend Snippet: PARylation primes HMGB1 for acetylation following rotenone treatment that is associated with decreased SIRT1 activity. A , graphical representation of post-translational modification induced by PARP1 that modulates SIRT1-HMGB1 interaction during ongoing DNA damage. B , determination of SIRT1 activity by fluorometric assay in SH-SY5Y cells following rotenone treatment in the presence or absence of PJ34 and compared with DMSO-treated controls. Results are expressed in arbitrary fluorescence units. C , immunoprecipitation analysis with anti-HMGB1 antibody to check the acetylation status of HMGB1 in the whole cell extracts of cells following rotenone treatment for 24 h and probing with pan-acetyl antibody. D and E , representative confocal images of cells stained with SIRT1 ( red ), HMGB1 ( green ) and DAPI ( blue ) following 4 ( D ) or 12 ( E ) h rotenone treatment. (scale bar = 10 μm). F , quantification of SIRT1 intensity at 12 h post rotenone treatment. (n = 100 cells for each quantification). G , representative confocal images of cells stained with SIRT1 (red), HMGB1 ( green ), and DAPI ( blue ) following 24 h rotenone treatment in the presence or absence of PJ34 or a combination of PJ34 and the SIRT1 inhibitor EX527. (scale bar = 10 μm). H , MTT assay of cells treated with rotenone for 24 h in the presence or absence of PJ34 or a combination of PJ34 and EX527. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test (∗∗ p < 0.01; ∗∗∗ p < 0.001; n = 3 biological replicates).

Article Snippet: Rabbit monoclonal antibody against PARP1 (#9532) was obtained from Cell Signaling Technology Inc. (CST).

Techniques: Activity Assay, Modification, Fluorescence, Immunoprecipitation, Staining, MTT Assay, Comparison