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


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    Structured Review

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

    (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.
    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

    (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).
    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

    (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.
    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

    (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).
    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

    (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).
    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

    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.
    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



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    Image Search Results


    (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.

    Journal: bioRxiv

    Article Title: The PIN1-p38-CtIP signaling axis protects stalled replication forks from deleterious degradation

    doi: 10.1101/2024.06.25.600562

    Figure Lengend 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.

    Article Snippet: For immunoblotting the following antibodies are used: mouse anti-Myc (9E10) 1:500, Thermo Fisher Scientific MA1-980; mouse anti-CtIP (D4) 1:250, Santa Cruz sc-271339; rabbit anti-CtIP (D76F7) 1:1000, Cell Signaling #9201; rabbit anti-pS276-CtIP 1:200 custom made with Eurogentec with synthetic phosphopeptides (KLH-coupled) corresponding to residues surrounding S276 (ETQGPMpSPLGDEL) ; mouse anti-Mre11 1:1000 Genetex #GTX70212; mouse anti-p38a 1:1000 Cell Signaling #9217; rabbit anti-p38a 1:1000 Cell Signaling #9218; rabbit anti-Phospho-p38 MAPK T180/Y182 Cell Signaling #9211; mouse anti-FLAG M2 1:1000, Sigma-Aldrich F1804; rabbit anti-Lamin B1 1:1000 ab16048; mouse anti-Tubulin 1:20’000 Sigma-Aldrich #T9026; rabbit anti-GFP 1:1000, Abcam ab290; mouse anti-GFP (B2) 1:500, Santa Cruz sc-9996; rabbit anti-Cyclin D1 1:1000, Cell Signaling #2922; rabbit anti-SMARCAL1 1:1000, Abcam ab154226; mouse anti-GAPDH 1:40’000, Millipore MAB374; mouse anti-BRCA1 (D9) 1:50, Santa Cruz sc-6954.

    Techniques: Sequencing, Modification, Immunoprecipitation, Transfection, Western Blot, Expressing, Whisker Assay, Standard Deviation

    (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).

    Journal: bioRxiv

    Article Title: The PIN1-p38-CtIP signaling axis protects stalled replication forks from deleterious degradation

    doi: 10.1101/2024.06.25.600562

    Figure Lengend 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).

    Article Snippet: For immunoblotting the following antibodies are used: mouse anti-Myc (9E10) 1:500, Thermo Fisher Scientific MA1-980; mouse anti-CtIP (D4) 1:250, Santa Cruz sc-271339; rabbit anti-CtIP (D76F7) 1:1000, Cell Signaling #9201; rabbit anti-pS276-CtIP 1:200 custom made with Eurogentec with synthetic phosphopeptides (KLH-coupled) corresponding to residues surrounding S276 (ETQGPMpSPLGDEL) ; mouse anti-Mre11 1:1000 Genetex #GTX70212; mouse anti-p38a 1:1000 Cell Signaling #9217; rabbit anti-p38a 1:1000 Cell Signaling #9218; rabbit anti-Phospho-p38 MAPK T180/Y182 Cell Signaling #9211; mouse anti-FLAG M2 1:1000, Sigma-Aldrich F1804; rabbit anti-Lamin B1 1:1000 ab16048; mouse anti-Tubulin 1:20’000 Sigma-Aldrich #T9026; rabbit anti-GFP 1:1000, Abcam ab290; mouse anti-GFP (B2) 1:500, Santa Cruz sc-9996; rabbit anti-Cyclin D1 1:1000, Cell Signaling #2922; rabbit anti-SMARCAL1 1:1000, Abcam ab154226; mouse anti-GAPDH 1:40’000, Millipore MAB374; mouse anti-BRCA1 (D9) 1:50, Santa Cruz sc-6954.

    Techniques: Transfection, Western Blot, Immunoprecipitation, Expressing, Mutagenesis, Whisker Assay, Standard Deviation

    (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.

    Journal: bioRxiv

    Article Title: The PIN1-p38-CtIP signaling axis protects stalled replication forks from deleterious degradation

    doi: 10.1101/2024.06.25.600562

    Figure Lengend 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.

    Article Snippet: For immunoblotting the following antibodies are used: mouse anti-Myc (9E10) 1:500, Thermo Fisher Scientific MA1-980; mouse anti-CtIP (D4) 1:250, Santa Cruz sc-271339; rabbit anti-CtIP (D76F7) 1:1000, Cell Signaling #9201; rabbit anti-pS276-CtIP 1:200 custom made with Eurogentec with synthetic phosphopeptides (KLH-coupled) corresponding to residues surrounding S276 (ETQGPMpSPLGDEL) ; mouse anti-Mre11 1:1000 Genetex #GTX70212; mouse anti-p38a 1:1000 Cell Signaling #9217; rabbit anti-p38a 1:1000 Cell Signaling #9218; rabbit anti-Phospho-p38 MAPK T180/Y182 Cell Signaling #9211; mouse anti-FLAG M2 1:1000, Sigma-Aldrich F1804; rabbit anti-Lamin B1 1:1000 ab16048; mouse anti-Tubulin 1:20’000 Sigma-Aldrich #T9026; rabbit anti-GFP 1:1000, Abcam ab290; mouse anti-GFP (B2) 1:500, Santa Cruz sc-9996; rabbit anti-Cyclin D1 1:1000, Cell Signaling #2922; rabbit anti-SMARCAL1 1:1000, Abcam ab154226; mouse anti-GAPDH 1:40’000, Millipore MAB374; mouse anti-BRCA1 (D9) 1:50, Santa Cruz sc-6954.

    Techniques: Expressing, Mutagenesis

    (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).

    Journal: bioRxiv

    Article Title: The PIN1-p38-CtIP signaling axis protects stalled replication forks from deleterious degradation

    doi: 10.1101/2024.06.25.600562

    Figure Lengend 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).

    Article Snippet: For immunoblotting the following antibodies are used: mouse anti-Myc (9E10) 1:500, Thermo Fisher Scientific MA1-980; mouse anti-CtIP (D4) 1:250, Santa Cruz sc-271339; rabbit anti-CtIP (D76F7) 1:1000, Cell Signaling #9201; rabbit anti-pS276-CtIP 1:200 custom made with Eurogentec with synthetic phosphopeptides (KLH-coupled) corresponding to residues surrounding S276 (ETQGPMpSPLGDEL) ; mouse anti-Mre11 1:1000 Genetex #GTX70212; mouse anti-p38a 1:1000 Cell Signaling #9217; rabbit anti-p38a 1:1000 Cell Signaling #9218; rabbit anti-Phospho-p38 MAPK T180/Y182 Cell Signaling #9211; mouse anti-FLAG M2 1:1000, Sigma-Aldrich F1804; rabbit anti-Lamin B1 1:1000 ab16048; mouse anti-Tubulin 1:20’000 Sigma-Aldrich #T9026; rabbit anti-GFP 1:1000, Abcam ab290; mouse anti-GFP (B2) 1:500, Santa Cruz sc-9996; rabbit anti-Cyclin D1 1:1000, Cell Signaling #2922; rabbit anti-SMARCAL1 1:1000, Abcam ab154226; mouse anti-GAPDH 1:40’000, Millipore MAB374; mouse anti-BRCA1 (D9) 1:50, Santa Cruz sc-6954.

    Techniques: Derivative Assay, Colony Assay, Standard Deviation

    (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).

    Journal: bioRxiv

    Article Title: The PIN1-p38-CtIP signaling axis protects stalled replication forks from deleterious degradation

    doi: 10.1101/2024.06.25.600562

    Figure Lengend 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).

    Article Snippet: For immunoblotting the following antibodies are used: mouse anti-Myc (9E10) 1:500, Thermo Fisher Scientific MA1-980; mouse anti-CtIP (D4) 1:250, Santa Cruz sc-271339; rabbit anti-CtIP (D76F7) 1:1000, Cell Signaling #9201; rabbit anti-pS276-CtIP 1:200 custom made with Eurogentec with synthetic phosphopeptides (KLH-coupled) corresponding to residues surrounding S276 (ETQGPMpSPLGDEL) ; mouse anti-Mre11 1:1000 Genetex #GTX70212; mouse anti-p38a 1:1000 Cell Signaling #9217; rabbit anti-p38a 1:1000 Cell Signaling #9218; rabbit anti-Phospho-p38 MAPK T180/Y182 Cell Signaling #9211; mouse anti-FLAG M2 1:1000, Sigma-Aldrich F1804; rabbit anti-Lamin B1 1:1000 ab16048; mouse anti-Tubulin 1:20’000 Sigma-Aldrich #T9026; rabbit anti-GFP 1:1000, Abcam ab290; mouse anti-GFP (B2) 1:500, Santa Cruz sc-9996; rabbit anti-Cyclin D1 1:1000, Cell Signaling #2922; rabbit anti-SMARCAL1 1:1000, Abcam ab154226; mouse anti-GAPDH 1:40’000, Millipore MAB374; mouse anti-BRCA1 (D9) 1:50, Santa Cruz sc-6954.

    Techniques: Colony Assay, Derivative Assay, Standard Deviation

    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.

    Journal: bioRxiv

    Article Title: The PIN1-p38-CtIP signaling axis protects stalled replication forks from deleterious degradation

    doi: 10.1101/2024.06.25.600562

    Figure Lengend 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.

    Article Snippet: For immunoblotting the following antibodies are used: mouse anti-Myc (9E10) 1:500, Thermo Fisher Scientific MA1-980; mouse anti-CtIP (D4) 1:250, Santa Cruz sc-271339; rabbit anti-CtIP (D76F7) 1:1000, Cell Signaling #9201; rabbit anti-pS276-CtIP 1:200 custom made with Eurogentec with synthetic phosphopeptides (KLH-coupled) corresponding to residues surrounding S276 (ETQGPMpSPLGDEL) ; mouse anti-Mre11 1:1000 Genetex #GTX70212; mouse anti-p38a 1:1000 Cell Signaling #9217; rabbit anti-p38a 1:1000 Cell Signaling #9218; rabbit anti-Phospho-p38 MAPK T180/Y182 Cell Signaling #9211; mouse anti-FLAG M2 1:1000, Sigma-Aldrich F1804; rabbit anti-Lamin B1 1:1000 ab16048; mouse anti-Tubulin 1:20’000 Sigma-Aldrich #T9026; rabbit anti-GFP 1:1000, Abcam ab290; mouse anti-GFP (B2) 1:500, Santa Cruz sc-9996; rabbit anti-Cyclin D1 1:1000, Cell Signaling #2922; rabbit anti-SMARCAL1 1:1000, Abcam ab154226; mouse anti-GAPDH 1:40’000, Millipore MAB374; mouse anti-BRCA1 (D9) 1:50, Santa Cruz sc-6954.

    Techniques: Binding Assay

    Investigation of phosphosites showing EGF-dependent up-regulation. ( A ) Scheme to filter peptides showing up-regulation by inhibitor treatment and summary of the numbers of the up-regulated phosphosites for each inhibitor ( B ) PCA of the log2-transformed abundance of phosphosites up-regulated by both 8 and 20 min of EGF incubation. ( C ) STRING-based protein network of proteins showing sanguinarine-dependent up-regulation of its phosphorylation. Proteins with at least one connection with other proteins are included. ( D ) Illustration of the p38 pathway activated by EGFR. The figure was created using BioRender.com. ( E ) Sequence motif enrichment analysis of 15 residues surrounding the regulated phosphosites. ( F ) Heatmap showing the log2-transformed phosphosite abundance changes induced by inhibitor treatment. See also Fig. S5 for the heatmap containing all 80 peptides. ( G ) Western blotting analysis using the cell lysates of Hela cells pre-treated with the inhibitors (500 nM) for 15 min followed by 20 min incubation with EGF (20 nM).

    Journal: Scientific Reports

    Article Title: Phosphoproteomic investigation of targets of protein phosphatases in EGFR signaling

    doi: 10.1038/s41598-024-58619-1

    Figure Lengend Snippet: Investigation of phosphosites showing EGF-dependent up-regulation. ( A ) Scheme to filter peptides showing up-regulation by inhibitor treatment and summary of the numbers of the up-regulated phosphosites for each inhibitor ( B ) PCA of the log2-transformed abundance of phosphosites up-regulated by both 8 and 20 min of EGF incubation. ( C ) STRING-based protein network of proteins showing sanguinarine-dependent up-regulation of its phosphorylation. Proteins with at least one connection with other proteins are included. ( D ) Illustration of the p38 pathway activated by EGFR. The figure was created using BioRender.com. ( E ) Sequence motif enrichment analysis of 15 residues surrounding the regulated phosphosites. ( F ) Heatmap showing the log2-transformed phosphosite abundance changes induced by inhibitor treatment. See also Fig. S5 for the heatmap containing all 80 peptides. ( G ) Western blotting analysis using the cell lysates of Hela cells pre-treated with the inhibitors (500 nM) for 15 min followed by 20 min incubation with EGF (20 nM).

    Article Snippet: Primary antibodies used were rabbit anti-EGFR (ab32198; Abcam), rabbit anti-phospho-EGFR (pY1045) (#2237; Cell Signaling Technology), mouse anti-phospho-EGFR (pY1068) (#2236; Cell Signaling Technology), rabbit anti-Erk1/2 (#4695; Cell Signaling Technology), mouse anti-phospho-Erk1/2 (T202/Y204) (#9106; Cell Signaling Technology), rabbit anti-Akt (#9272; Cell Signaling Technology) rabbit anti-phospho-Akt (S473) (#9271; Cell Signaling Technology), rabbit anti-p38 MAPK (#9212; Cell Signaling Technology), rabbit anti-phospho p38 MAPK (T180/Y182) (#9211; Cell Signaling Technology) and mouse anti-GAPDH (Abcam).

    Techniques: Transformation Assay, Incubation, Sequencing, Western Blot

    MS-based phosphoproteomics of EGF signaling with treatment of PP2C inhibitor sanguinarine in presence of p38 inhibitor BIRB796. ( A ) Schematic illustration of MS-based phosphoproteomics sample preparation. The figure was created using BioRender.com. ( B ) STRING-based protein network of proteins showing sanguinarine-dependent up-regulation of its phosphorylation. ( C ) Heatmap showing the log2-transformed phosphosite abundance changes induced by inhibitor treatment.

    Journal: Scientific Reports

    Article Title: Phosphoproteomic investigation of targets of protein phosphatases in EGFR signaling

    doi: 10.1038/s41598-024-58619-1

    Figure Lengend Snippet: MS-based phosphoproteomics of EGF signaling with treatment of PP2C inhibitor sanguinarine in presence of p38 inhibitor BIRB796. ( A ) Schematic illustration of MS-based phosphoproteomics sample preparation. The figure was created using BioRender.com. ( B ) STRING-based protein network of proteins showing sanguinarine-dependent up-regulation of its phosphorylation. ( C ) Heatmap showing the log2-transformed phosphosite abundance changes induced by inhibitor treatment.

    Article Snippet: Primary antibodies used were rabbit anti-EGFR (ab32198; Abcam), rabbit anti-phospho-EGFR (pY1045) (#2237; Cell Signaling Technology), mouse anti-phospho-EGFR (pY1068) (#2236; Cell Signaling Technology), rabbit anti-Erk1/2 (#4695; Cell Signaling Technology), mouse anti-phospho-Erk1/2 (T202/Y204) (#9106; Cell Signaling Technology), rabbit anti-Akt (#9272; Cell Signaling Technology) rabbit anti-phospho-Akt (S473) (#9271; Cell Signaling Technology), rabbit anti-p38 MAPK (#9212; Cell Signaling Technology), rabbit anti-phospho p38 MAPK (T180/Y182) (#9211; Cell Signaling Technology) and mouse anti-GAPDH (Abcam).

    Techniques: Sample Prep, Transformation Assay

    P38 is required for ammonia-enhanced WSSV infection. ( A ) Suppression of ammonia-enhanced WSSV infection by p38 inhibition. Shrimp were maintained in normal seawater or subjected to ammonia stress. The inhibitors were then administered 30 min later. WSSV infection was performed after 12 h. VP28 levels and viral loads were measured in the gills. ( B ) Suppression of ammonia-induced Hsf1 activation by p38 inhibition. Shrimp were maintained in normal seawater or subjected to ammonia stress. The inhibitor was then administered 30 min later. The nuclear levels of Hsf1 in the gills were determined 2 h later. ( C ) Suppression of ammonia-mediated changes in gene expression caused by p38 inhibition. Shrimp were maintained in normal seawater or subjected to ammonia stress. The inhibitors were then administered 30 min later. WSSV infection was performed after 12 h. Gene expression and protein production levels were determined 24 h after infection. ( D ) Suppression of ammonia-reduced nuclear levels of Dorsal and Stat by p38 inhibition. Immunocytochemical analyses were performed 24 h after WSSV infection using the indicated antibodies. Scale bar = 10 μm. ( E ) Suppression of ammonia-reduced Dorsal/ MjVago-L transcription and Stat/ MjFicolin transcription by p38 inhibition. ChIP assays were performed 24 h after WSSV infection. All bar charts show the mean ± SD from three replicates. ∗∗∗ P < 0.001, ∗∗0.001 < P < .01, and ∗ P < 0.05, as determined using Student’s t -test. Images are representative of three or four replicates. At least five shrimp were used to prepare the samples.

    Journal: mBio

    Article Title: Ammonia stress-induced heat shock factor 1 enhances white spot syndrome virus infection by targeting the interferon-like system in shrimp

    doi: 10.1128/mbio.03136-23

    Figure Lengend Snippet: P38 is required for ammonia-enhanced WSSV infection. ( A ) Suppression of ammonia-enhanced WSSV infection by p38 inhibition. Shrimp were maintained in normal seawater or subjected to ammonia stress. The inhibitors were then administered 30 min later. WSSV infection was performed after 12 h. VP28 levels and viral loads were measured in the gills. ( B ) Suppression of ammonia-induced Hsf1 activation by p38 inhibition. Shrimp were maintained in normal seawater or subjected to ammonia stress. The inhibitor was then administered 30 min later. The nuclear levels of Hsf1 in the gills were determined 2 h later. ( C ) Suppression of ammonia-mediated changes in gene expression caused by p38 inhibition. Shrimp were maintained in normal seawater or subjected to ammonia stress. The inhibitors were then administered 30 min later. WSSV infection was performed after 12 h. Gene expression and protein production levels were determined 24 h after infection. ( D ) Suppression of ammonia-reduced nuclear levels of Dorsal and Stat by p38 inhibition. Immunocytochemical analyses were performed 24 h after WSSV infection using the indicated antibodies. Scale bar = 10 μm. ( E ) Suppression of ammonia-reduced Dorsal/ MjVago-L transcription and Stat/ MjFicolin transcription by p38 inhibition. ChIP assays were performed 24 h after WSSV infection. All bar charts show the mean ± SD from three replicates. ∗∗∗ P < 0.001, ∗∗0.001 < P < .01, and ∗ P < 0.05, as determined using Student’s t -test. Images are representative of three or four replicates. At least five shrimp were used to prepare the samples.

    Article Snippet: Cross-reacting rabbit anti-human histone H3 polyclonal antibodies (ProteinTech, Wuhan, China; 17168-1-AP) were used to detect shrimp H3 expression at 1:1,000 dilution, and anti-phospho-p38 MAPK-T180/Y182 rabbit polyclonal antibodies (ABclonal, Woburn, MA, USA; AP0526) were used to analyze shrimp p38 phosphorylation at 1:1,000 dilution.

    Techniques: Infection, Inhibition, Activation Assay, Expressing

    Involvement of calcium influx in ammonia-enhanced WSSV infection. ( A ) Induction of calcium influx in gills by ammonia stress treatment. Shrimp were maintained in normal seawater or subjected to ammonia stress treatment. The calcium chelator BAPTA-AM was administered 30 min later. After another 30 min, the tissues were stained with the calcium fluorescence probe Fluo-3AM and observed under a confocal microscope (left panel). The intracellular calcium concentration was determined using a calcium colorimetric assay kit (right panel). ( B ) Suppression of ammonia-induced p38 activation by calcium chelation. Shrimp were maintained in normal seawater or subjected to ammonia stress treatment. BAPTA-AM or the control treatment was administered 30 min later. The p38 levels in the gills were determined 12 h later. ( C ) Suppression of ammonia-induced gene expression variation by calcium chelation. Shrimp were maintained in normal seawater or subjected to ammonia stress treatment. BAPTA-AM or the control treatment was administered 30 min later. Gene expression levels were determined another 12 h later, while protein production levels were determined another 24 h later. ( D ) Suppression of ammonia-enhanced WSSV infection by calcium chelation. Shrimp were maintained in normal seawater or subjected to ammonia stress treatment. BAPTA-AM or the control treatment was administered 30 min later. WSSV infection was performed another 12 h later. The VP28 levels in the gills were determined 24 h after infection. ( E ) Working model of this study. Ammonia stress induces the production of Hsf1, leading to calcium influx, which activates p38 and Hsf1. Activated Hsf1 induces the production of Cactus and Socs2, which target the shrimp interferon-like system at two nodes, namely, Dorsal and Stat. By suppressing shrimp antiviral immunity through inhibiting the production and function of MjVago-L, ammonia stress enhances WSSV infection. All bar charts show the mean ± SD from three replicates. ∗∗∗ P < 0.001, ∗∗0.001 < P < .01, and ∗ P < 0.05, as determined using Student’s t -test. Images are representative of three replicates. At least five shrimp were used to prepare the samples.

    Journal: mBio

    Article Title: Ammonia stress-induced heat shock factor 1 enhances white spot syndrome virus infection by targeting the interferon-like system in shrimp

    doi: 10.1128/mbio.03136-23

    Figure Lengend Snippet: Involvement of calcium influx in ammonia-enhanced WSSV infection. ( A ) Induction of calcium influx in gills by ammonia stress treatment. Shrimp were maintained in normal seawater or subjected to ammonia stress treatment. The calcium chelator BAPTA-AM was administered 30 min later. After another 30 min, the tissues were stained with the calcium fluorescence probe Fluo-3AM and observed under a confocal microscope (left panel). The intracellular calcium concentration was determined using a calcium colorimetric assay kit (right panel). ( B ) Suppression of ammonia-induced p38 activation by calcium chelation. Shrimp were maintained in normal seawater or subjected to ammonia stress treatment. BAPTA-AM or the control treatment was administered 30 min later. The p38 levels in the gills were determined 12 h later. ( C ) Suppression of ammonia-induced gene expression variation by calcium chelation. Shrimp were maintained in normal seawater or subjected to ammonia stress treatment. BAPTA-AM or the control treatment was administered 30 min later. Gene expression levels were determined another 12 h later, while protein production levels were determined another 24 h later. ( D ) Suppression of ammonia-enhanced WSSV infection by calcium chelation. Shrimp were maintained in normal seawater or subjected to ammonia stress treatment. BAPTA-AM or the control treatment was administered 30 min later. WSSV infection was performed another 12 h later. The VP28 levels in the gills were determined 24 h after infection. ( E ) Working model of this study. Ammonia stress induces the production of Hsf1, leading to calcium influx, which activates p38 and Hsf1. Activated Hsf1 induces the production of Cactus and Socs2, which target the shrimp interferon-like system at two nodes, namely, Dorsal and Stat. By suppressing shrimp antiviral immunity through inhibiting the production and function of MjVago-L, ammonia stress enhances WSSV infection. All bar charts show the mean ± SD from three replicates. ∗∗∗ P < 0.001, ∗∗0.001 < P < .01, and ∗ P < 0.05, as determined using Student’s t -test. Images are representative of three replicates. At least five shrimp were used to prepare the samples.

    Article Snippet: Cross-reacting rabbit anti-human histone H3 polyclonal antibodies (ProteinTech, Wuhan, China; 17168-1-AP) were used to detect shrimp H3 expression at 1:1,000 dilution, and anti-phospho-p38 MAPK-T180/Y182 rabbit polyclonal antibodies (ABclonal, Woburn, MA, USA; AP0526) were used to analyze shrimp p38 phosphorylation at 1:1,000 dilution.

    Techniques: Infection, Staining, Fluorescence, Microscopy, Concentration Assay, Calcium Colorimetric Assay, Activation Assay, Expressing

    The BMP7/SMAD1/YAP and BMP7/p38 MAPK pathways mediate omentin-1-induced cardiomyocyte (CM) cell cycle arrest and metabolic maturation. a Heatmap of gene expression profiles in control CMs, BMP7- and OMT-cotreated CMs, and BMP7-treated CMs. b Differential expression analyses of sarcomere, metabolism maturation, and cell cycle arrest genes. The purple-green color scale indicates the differential expression fold change. c Gene Ontology (GO) enrichment analysis of differentially expressed genes in green clusters. d Western blot analysis showing time-dependent changes in BMP7 signaling downstream proteins, including SMAD1, p-SMAD1, YAP, p-YAP, p38 MAPK, and p-p38MAPK (left). Primary CMs were pretreated with an empty virus control (EV) and omentin-1 overexpression (OE) virus and then stimulated with BMP7 protein (100 ng/ml). The phosphorylation levels were determined by corresponding band intensity measurements, and the values were determined relative to β-actin signals. e Western blot analysis detected p-SMAD1, p-YAP, and p-p38 MAPK proteins in the cytoplasm and nuclear fraction in EV/omentin-1-OE CMs with BMP7 stimulation. f Immunofluorescence of primary rat CM proliferation (pH3) in response to BMP7, BMP7 + YAP inhibitor (verteporfin), and BMP7 + MAPK inhibitor (SB203580); scale bar, 20 μm. g Mitochondrial stress assay demonstrating the mitochondrial respiration and proton leak in four primary rat CM groups; the PBS control, BMP7, BMP7 + MAPK inhibitor (SB203580), and BMP7 + YAP inhibitor (verteporfin) groups. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05 by unpaired Student's t test

    Journal: Cellular and Molecular Life Sciences: CMLS

    Article Title: Omentin-1 drives cardiomyocyte cell cycle arrest and metabolic maturation by interacting with BMP7

    doi: 10.1007/s00018-023-04829-1

    Figure Lengend Snippet: The BMP7/SMAD1/YAP and BMP7/p38 MAPK pathways mediate omentin-1-induced cardiomyocyte (CM) cell cycle arrest and metabolic maturation. a Heatmap of gene expression profiles in control CMs, BMP7- and OMT-cotreated CMs, and BMP7-treated CMs. b Differential expression analyses of sarcomere, metabolism maturation, and cell cycle arrest genes. The purple-green color scale indicates the differential expression fold change. c Gene Ontology (GO) enrichment analysis of differentially expressed genes in green clusters. d Western blot analysis showing time-dependent changes in BMP7 signaling downstream proteins, including SMAD1, p-SMAD1, YAP, p-YAP, p38 MAPK, and p-p38MAPK (left). Primary CMs were pretreated with an empty virus control (EV) and omentin-1 overexpression (OE) virus and then stimulated with BMP7 protein (100 ng/ml). The phosphorylation levels were determined by corresponding band intensity measurements, and the values were determined relative to β-actin signals. e Western blot analysis detected p-SMAD1, p-YAP, and p-p38 MAPK proteins in the cytoplasm and nuclear fraction in EV/omentin-1-OE CMs with BMP7 stimulation. f Immunofluorescence of primary rat CM proliferation (pH3) in response to BMP7, BMP7 + YAP inhibitor (verteporfin), and BMP7 + MAPK inhibitor (SB203580); scale bar, 20 μm. g Mitochondrial stress assay demonstrating the mitochondrial respiration and proton leak in four primary rat CM groups; the PBS control, BMP7, BMP7 + MAPK inhibitor (SB203580), and BMP7 + YAP inhibitor (verteporfin) groups. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05 by unpaired Student's t test

    Article Snippet: The membranes were blocked with 5% fetal calf serum (FCS) in PBS at room temperature for 1 h and then incubated overnight at 4 °C with the following primary antibodies: anti-Omentin-1 (GeneTex, GTX32687, 1:1000 dilution), anti-CDK2, anti-CDK4, anti-CDK6, anti-cyclin D1 (CST, all 1:1000, Cell Cycle Regulation Antibody Sampler Kit #9932), anti-c-Myc (CST, 18,583, 1:1000 dilution), anti-BMP7 (Proteintech, 12,221–1-AP, 1:1000 dilution), anti-Flag (Sigma, SAB1306078, 1:1000 dilution), anti-YAP (CST, 14,074, 1:1000 dilution), anti-YAP-s127 (CST, 4911, 1:1000 dilution), anti-phospho-Smad1-S206 rabbit (Abclonal, AP0295, 1:1000 dilution), anti-SMAD1 (Abclonal, A1101, 1:1000 dilution), anti-phospho-p38 MAPK-T180/Y182 rabbit (Abclonal, AP0526, 1:1000 dilution), and anti-p38 MAPK (Abclonal, A14401, 1:1000 dilution).

    Techniques: Expressing, Western Blot, Virus, Over Expression, Immunofluorescence

    Model of BMP7–omentin-1 in cardiomyocyte (CM) cell cycle arrest and metabolic maturation. The expression of omentin-1 is upregulated during postnatal heart development. Omentin-1 binds to BMP7 extracellularly, prevents BMP7 from binding to its receptor ActRIIB, and blocks the SMAD1/YAP and p38 MAPK pathways, which induces CM maturation and cell cycle arrest. BMP7, bone morphogenetic protein 7; SMAD1, mothers against DPP homolog 1; p38 MAPK, p38 mitogen-activated protein kinase; YAP, yes-associated protein; ActRIIB, activin type II receptor B

    Journal: Cellular and Molecular Life Sciences: CMLS

    Article Title: Omentin-1 drives cardiomyocyte cell cycle arrest and metabolic maturation by interacting with BMP7

    doi: 10.1007/s00018-023-04829-1

    Figure Lengend Snippet: Model of BMP7–omentin-1 in cardiomyocyte (CM) cell cycle arrest and metabolic maturation. The expression of omentin-1 is upregulated during postnatal heart development. Omentin-1 binds to BMP7 extracellularly, prevents BMP7 from binding to its receptor ActRIIB, and blocks the SMAD1/YAP and p38 MAPK pathways, which induces CM maturation and cell cycle arrest. BMP7, bone morphogenetic protein 7; SMAD1, mothers against DPP homolog 1; p38 MAPK, p38 mitogen-activated protein kinase; YAP, yes-associated protein; ActRIIB, activin type II receptor B

    Article Snippet: The membranes were blocked with 5% fetal calf serum (FCS) in PBS at room temperature for 1 h and then incubated overnight at 4 °C with the following primary antibodies: anti-Omentin-1 (GeneTex, GTX32687, 1:1000 dilution), anti-CDK2, anti-CDK4, anti-CDK6, anti-cyclin D1 (CST, all 1:1000, Cell Cycle Regulation Antibody Sampler Kit #9932), anti-c-Myc (CST, 18,583, 1:1000 dilution), anti-BMP7 (Proteintech, 12,221–1-AP, 1:1000 dilution), anti-Flag (Sigma, SAB1306078, 1:1000 dilution), anti-YAP (CST, 14,074, 1:1000 dilution), anti-YAP-s127 (CST, 4911, 1:1000 dilution), anti-phospho-Smad1-S206 rabbit (Abclonal, AP0295, 1:1000 dilution), anti-SMAD1 (Abclonal, A1101, 1:1000 dilution), anti-phospho-p38 MAPK-T180/Y182 rabbit (Abclonal, AP0526, 1:1000 dilution), and anti-p38 MAPK (Abclonal, A14401, 1:1000 dilution).

    Techniques: Expressing, Binding Assay