Journal: Science Advances
Article Title: PARP1 stabilizes FOXN3 to suppress pulmonary fibrosis through p38-related feedback regulation
doi: 10.1126/sciadv.ady1681
Figure Lengend Snippet: ( A ) Affinity-purified immunoprecipitates from HEK293T cells via anti-Flag immunoprecipitation (IP) were analyzed by mass spectrometry to identify unique peptides associated with Flag-tagged FOXN3. The sequences of the PARP1 peptides linked to FOXN3 are presented. ( B ) A co-IP assay was carried out in A549 cells to examine the endogenous association between FOXN3 and PARP1. ( C ) An anti-Flag co-IP assay was performed in A549 cells stably expressing Flag-tagged FOXN3 to assess the association between FOXN3 and PARP1 upon TGF-β treatment (10 ng/ml) at the indicated time points [0, 16, 24, and 48 hours (hr)]. IB, immunoblot. The cells were treated with MG-132 (20 μM, 4 hours) before collection. ( D ) An immunofluorescence assay was performed in A549 cells to assess the colocalization of FOXN3 and PARP1. Scale bar, 10 μm. ( E ) A deletion-mapping assay was performed in HEK293T cells to define the region within FOXN3 associated with PARP1. Flag-tagged FOXN3 WT or its various mutants were cotransfected with HA-tagged PARP1 into HEK293T cells (ΔNTD: Δ1-113aa; ΔForkhead: Δ114-199aa; ΔCTD: Δ200-490aa). ( F ) An in vitro GST pull-down assay was performed to examine the direct interaction between bacterially expressed GST-tagged FOXN3 and His-tagged PARP1 purified from Sf9 insect cells. ( G ) Venn diagrams illustrating the overlapping genes cotargeted by FOXN3 and PARP1 in A549 cells, as determined via CUT&Tag data analysis. ( H ) The normalized distribution of reads for the FOXN3 and PARP1 peaks at the transcription start site (TSS) is presented. ( I ) The binding profiles of FOXN3 and PARP1 at representative Smad response genes in A549 cells were analyzed via CUT&Tag data. The blotting data for (B), (C), (E), and (F) were quantified as the means from three independent experiments using ImageJ software. The data for (C) and (E) were analyzed using one-way ANOVA. ** P < 0.01 and *** P < 0.001.
Article Snippet: The PARP1 protein purified from Sf9 insect cells was obtained from MedChemExpress (no. HY- P74652 ).
Techniques: Affinity Purification, Immunoprecipitation, Mass Spectrometry, Co-Immunoprecipitation Assay, Stable Transfection, Expressing, Western Blot, Immunofluorescence, Mapping Assay, In Vitro, Pull Down Assay, Purification, Binding Assay, Software
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![( A ) Affinity-purified immunoprecipitates from HEK293T cells via anti-Flag immunoprecipitation (IP) were analyzed by mass spectrometry to identify unique peptides associated with Flag-tagged FOXN3. The sequences of the PARP1 peptides linked to FOXN3 are presented. ( B ) A co-IP assay was carried out in A549 cells to examine the endogenous association between FOXN3 and PARP1. ( C ) An anti-Flag co-IP assay was performed in A549 cells stably expressing Flag-tagged FOXN3 to assess the association between FOXN3 and PARP1 upon TGF-β treatment (10 ng/ml) at the indicated time points [0, 16, 24, and 48 hours (hr)]. IB, immunoblot. The cells were treated with MG-132 (20 μM, 4 hours) before collection. ( D ) An immunofluorescence assay was performed in A549 cells to assess the colocalization of FOXN3 and PARP1. Scale bar, 10 μm. ( E ) A deletion-mapping assay was performed in HEK293T cells to define the region within FOXN3 associated with PARP1. Flag-tagged FOXN3 WT or its various mutants were cotransfected with HA-tagged PARP1 into HEK293T cells (ΔNTD: Δ1-113aa; ΔForkhead: Δ114-199aa; ΔCTD: Δ200-490aa). ( F ) An in vitro GST pull-down assay was performed to examine the direct interaction between bacterially expressed GST-tagged FOXN3 and His-tagged PARP1 purified from <t>Sf9</t> insect cells. ( G ) Venn diagrams illustrating the overlapping genes cotargeted by FOXN3 and PARP1 in A549 cells, as determined via CUT&Tag data analysis. ( H ) The normalized distribution of reads for the FOXN3 and PARP1 peaks at the transcription start site (TSS) is presented. ( I ) The binding profiles of FOXN3 and PARP1 at representative Smad response genes in A549 cells were analyzed via CUT&Tag data. The blotting data for (B), (C), (E), and (F) were quantified as the means from three independent experiments using ImageJ software. The data for (C) and (E) were analyzed using one-way ANOVA. ** P < 0.01 and *** P < 0.001.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_8542/pmc12758542/pmc12758542__sciadv.ady1681-f1.jpg)