rabbit anti phospho p38 mapk t180 y182 cell signaling (Cell Signaling Technology Inc)

Structured Review

Rabbit Anti Phospho P38 Mapk T180 Y182 Cell Signaling, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/rabbit anti phospho p38 mapk t180 y182 cell signaling/product/Cell Signaling Technology Inc
Average 86 stars, based on 1 article reviews
Images
1) Product Images from "The PIN1-p38-CtIP signaling axis protects stalled replication forks from deleterious degradation"
Article Title: The PIN1-p38-CtIP signaling axis protects stalled replication forks from deleterious degradation
Journal: bioRxiv
doi: 10.1101/2024.06.25.600562

Figure Legend Snippet: (Related to ). (A) Multiple sequence alignment of the CtIP region containing S276. The full consensus sequence for p38α substrates is shown below (modified from Johnson et al., 2023 ). (B) Immunoprecipitation of endogenous CtIP from HEK293T cells transfected with Myc-p38α. Whole-cell lysates (input) and immunoprecipitates were analyzed by western blotting using specific antibodies. The * indicates an unspecific band. (C) GFP-Trap of U2OS cells inducibly expressing GFP-CtIP and treated with HU (2 mM, 4h). Where indicated, cells were treated with the p38α PROTAC NR-11c (1 µM, 24h before HU). Whole-cell lysates (input) and immunoprecipitates were analyzed by western blotting using specific antibodies. Densiometric quantification of CtIP-pS276 band in the GFP-Trap is shown (% indicate CtIP-pS276 band intensity vs CtIP band intensity). (D) Fork degradation was evaluated upon HU treatment in U2OS cells depleted of either endogenous CtIP or p38α. Box and whisker plots of IdU/CldU-tract length ratios for individual replication forks are shown. Numbers indicated above the individual plots represent the mean ratios ± standard deviation. Schematics of the CldU/IdU pulse-labelling protocol are shown (top). Western blotting of lysates from the same experiment is shown below. (E) Western blotting of lysates from cells used in . (F) Western blotting of lysates from wild-type mouse embryonic fibroblasts (MEFs) and Pin1 -/- MEFs.
Techniques Used: Sequencing, Modification, Immunoprecipitation, Transfection, Western Blot, Expressing, Whisker Assay, Standard Deviation

Figure Legend Snippet: (A) Myc-Trap of HEK293T cells transfected with Myc-p38α. Whole-cell lysates (input) and immunoprecipitates were analyzed by western blotting using specific antibodies. (B) Immunoprecipitation (IP) of CtIP-pS276 from U2OS cells inducibly expressing GFP-CtIP either mock-treated or treated with HU (2 mM, 4h). Where indicated, cells were treated with the p38α inhibitor PH-797804 (1 µM, 24h before HU). Whole-cell lysates (input) and immunoprecipitates were analyzed by western blotting using specific antibodies. Densiometric quantification of CtIP band in the IP is shown (% indicates CtIP band intensity vs IgG band intensity). (C) GFP-Trap of HEK293T cells co-transfected with GFP-PIN1 and indicated FLAG-CtIP variants. 24h post-transfection, cells were either mock-treated or treated with the p38α inhibitor PH-797804 (1 µM) for 24h. Whole-cell lysates (input) and immunoprecipitates were analyzed by western blotting using specific antibodies. (D) Fork degradation was evaluated upon HU treatment in U2OS cells either treated with the p38α inhibitor PH-797804 (1 µM, 24h before HU) or with the p38α PROTAC NR-11c (1 µM, 24h before HU). Western blotting of lysates from the same experiment is shown below. (E) Fork degradation was evaluated upon HU treatment in U2OS cells inducibly expressing siCtIP-resistant GFP-CtIP wt or S276A/P277A trans -locked mutant and depleted of endogenous CtIP alone, or co-depleted of CtIP and p38α. (F) Fork degradation was evaluated upon HU treatment in wild-type mouse embryonic fibroblasts (MEFs) and Pin1 -/- MEFs, pre-treated either for 24h with the p38α inhibitor PH-797804 (1 µM) or for 1h with the PIN1 inhibitor KPT-6566 (10 µM). (D-F) Box and whisker plots of IdU/CldU-tract length ratios for individual replication forks are shown. Numbers indicated above the individual plots represent the mean ratios ± standard deviation. Schematics of the CldU/IdU pulse-labelling protocol are shown (top).
Techniques Used: Transfection, Western Blot, Immunoprecipitation, Expressing, Mutagenesis, Whisker Assay, Standard Deviation

Figure Legend Snippet: (A) CtIP SIRF assay in U2OS cells pulsed-labelled with EdU (25 µM) for 10 min followed by treatment with HU (2mM) for 4h. Where indicated cells were treated with the PIN1 inhibitor KPT-6566 (10 µM, 1h before EdU labelling). (B) CtIP SIRF assay in U2OS cells pulsed-labelled with EdU (25 µM) for 10 min followed by treatment with HU (2mM) for 4h. Where indicated cells were treated with the p38α inhibitor PH-797804 (1 µM, 24h before EdU labelling). (C) GFP-CtIP SIRF assay in U2OS cells inducibly expressing siCtIP-resistant GFP-CtIP wt, S276A or S276A/P277A trans -locked mutant and depleted of endogenous CtIP. Cells were pulsed-labelled with EdU (25 µM) for 10 min followed by treatment with HU (2mM) for 4h. (A-C) Dot plots show the number of PLA foci and the median from at least 120 EdU-positive cells. Representative images are shown on top of each figure. Scale bars, 10 μm.
Techniques Used: Expressing, Mutagenesis

Figure Legend Snippet: (A) CtIP SIRF assay in KB1P-derived Trp53 -/- ; Brca1 -/- and Trp53 -/- ; Brca1 -/- ; H2afx -/- cells, either mock-treated or treated with the PIN1 inhibitor KPT-6566 (10 µM) for 1h, or with the p38α inhibitor PH-797804 (1 µM) for 24h. Cells were pulse-labelled with EdU (25 µM) for 10 min followed by treatment with HU (8 mM) alone or in combination with the PIN1 or p38α inhibitors for 6h. Dot plots show the number of PLA foci and the median from at least 150 EdU-positive cells. Representative images are shown on the right. Scale bars, 10 μm. (B) Colony formation assay was performed in same cells as in (A), either mock-treated or treated with the PIN1 inhibitor KPT-6566 (2.5 μM) and with the PARP inhibitor Olaparib (75 nM) for 10 days. (C) Colony formation assay was performed in same cells as in (A), either mock-treated or treated with the p38α inhibitor PH-797804 (10 μM) and with the PARP inhibitor Olaparib (75 nM) for 10 days. (B and C) Plotted values are mean ± standard deviation of three biological replicates. Representative images are shown (top).
Techniques Used: Derivative Assay, Colony Assay, Standard Deviation

Figure Legend Snippet: (Related to ). (A) Colony formation assay was performed in KB1P-derived Trp53 -/- ; Brca1 -/- and Trp53 -/- ; Brca1 -/- ; H2afx -/- cells, either mock-treated or treated with the PIN1 inhibitor KPT-6566 (7.5 μM) and HU (80 μM) for 10 days. (B) Colony formation assay was performed in same cells as in (A), either mock-treated or treated with the p38α inhibitor PH-797804 (10 μM) and HU (80 μM) for 10 days. (A and B) Plotted values are mean ± standard deviation of three biological replicates. Representative images are shown (top).
Techniques Used: Colony Assay, Derivative Assay, Standard Deviation

Figure Legend Snippet: During unperturbed S-phase, CDK2-mediated phosphorylation of T315 promotes PIN1 binding to CtIP. In response to replication stress, p38α kinase phosphorylates CtIP at S276. Subsequently, PIN1 catalyzes the cis -to- trans isomerization of the pS276-P277 peptide bond, ensuring accumulation of CtIP at stalled forks. Ultimately, this phosphorylation-isomerization cascade promotes CtIP-dependent protection of nascent DNA from DNA2-mediated nucleolytic processing, thereby maintaining of genome stability.
Techniques Used: Binding Assay