cer1 flag plasmids  (Sino Biological)


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    Name:
    CER1 cDNA ORF Clone Mouse C DYKDDDDK tag
    Description:
    Full length Clone DNA of Mouse cerberus1homolog Xenopuslaevis with C terminal Flag tag
    Catalog Number:
    MG51161-CF
    Price:
    195.0
    Category:
    cDNA Clone
    Applications:
    Stable or Transient mammalian expression
    Size:
    1Unit
    Product Aliases:
    cer-1 cDNA ORF Clone Mouse, Cerl cDNA ORF Clone Mouse, Cerl1 cDNA ORF Clone Mouse, Cerr1 cDNA ORF Clone Mouse
    Molecule Name:
    CER1,Cerr1,
    Buy from Supplier


    Structured Review

    Sino Biological cer1 flag plasmids
    CER1 cDNA ORF Clone Mouse C DYKDDDDK tag
    Full length Clone DNA of Mouse cerberus1homolog Xenopuslaevis with C terminal Flag tag
    https://www.bioz.com/result/cer1 flag plasmids/product/Sino Biological
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cer1 flag plasmids - by Bioz Stars, 2021-05
    86/100 stars

    Images

    1) Product Images from "ISM1 regulates NODAL signaling and asymmetric organ morphogenesis during development"

    Article Title: ISM1 regulates NODAL signaling and asymmetric organ morphogenesis during development

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201801081

    The inhibitory effects of ISM1, LEFTY1, and CER1 on NODAL signaling. (A) Serum-starved HEK293T-CRIPTO cells treated with 100 ng/ml rNODAL in the presence of mock, LEFTY1, ISM1, or CER1 CM at indicated dilutions. WCL samples were subjected to Western blotting analyses for pSMAD2. Total SMAD2/3 and β-actin were used as internal controls. The protein levels of LEFTY1, ISM1, and CER1 representing their relative concentrations in the CMs were analyzed by Western blotting using Flag antibodies. (B) Quantification of the intensity of pSMAD2 relative to SMAD2/3 in three biological replicates. Data represent mean ± SEM. The unpaired, two-tailed t test was used for statistical analyses. Statistically significant P values are indicated. (C) Dual luciferase reporter assay to determine the SMAD2/FOXH1 transcriptional activity in HEK293T-CRIPTO cells treated by 100 ng/ml rNODAL for 24 h, together with CM of mock, FLAG-LEFTY1, FLAG-ISM1, or FLAG-CER1 at the indicated dilutions. Data represent mean ± SEM of four independent experiments. P values of statistical analyses using unpaired, two-tailed t test are indicated in each individual experimental group.
    Figure Legend Snippet: The inhibitory effects of ISM1, LEFTY1, and CER1 on NODAL signaling. (A) Serum-starved HEK293T-CRIPTO cells treated with 100 ng/ml rNODAL in the presence of mock, LEFTY1, ISM1, or CER1 CM at indicated dilutions. WCL samples were subjected to Western blotting analyses for pSMAD2. Total SMAD2/3 and β-actin were used as internal controls. The protein levels of LEFTY1, ISM1, and CER1 representing their relative concentrations in the CMs were analyzed by Western blotting using Flag antibodies. (B) Quantification of the intensity of pSMAD2 relative to SMAD2/3 in three biological replicates. Data represent mean ± SEM. The unpaired, two-tailed t test was used for statistical analyses. Statistically significant P values are indicated. (C) Dual luciferase reporter assay to determine the SMAD2/FOXH1 transcriptional activity in HEK293T-CRIPTO cells treated by 100 ng/ml rNODAL for 24 h, together with CM of mock, FLAG-LEFTY1, FLAG-ISM1, or FLAG-CER1 at the indicated dilutions. Data represent mean ± SEM of four independent experiments. P values of statistical analyses using unpaired, two-tailed t test are indicated in each individual experimental group.

    Techniques Used: Western Blot, Two Tailed Test, Luciferase, Reporter Assay, Activity Assay

    AMOP domain is required for the function of ISM1 as an antagonist of NODAL signaling. (A) Diagram of domain-deleted ISM1 constructs lacking either TSR1 or AMOP (ΔTSR1 and ΔAMOP, respectively). The N -glycosylation sites at positions N39 and N282 are indicated. N39 is located in the N terminus, 10 amino acids downstream from the signal peptide (amino acids 1–29), and N282 is located in the region between TSR1 and AMOP domains (amino acids 260–285). (B) Western blot of ISM1 in WCL and CM samples of HEK293T cells transiently transfected with wild-type ISM1, ΔTSR1-ISM1, or ΔAMOP-ISM1. Samples were digested with PNGase F or Endo H before being subjected to Western blotting. (C) Mapping of NODAL-interacting domain of ISM1. HEK293T cells were transiently transfected with FLAG-NODAL together with either wild-type ISM1 or domain-deleted ISM1 mutants. CM samples were immunoprecipitated (IP) with anti-FLAG or anti-ISM1 antibodies and subjected to Western blotting with anti-ISM1 and anti-FLAG antibodies. (D) Mapping of ACVR1B ECD -interacting domain of ISM1. HEK293T cells transiently expressing ACVR1B ECD were cotransfected with wild-type ISM1 or domain-deleted ISM1 mutants. ACVR2B ECD were also cotransfected with ACVR1B ECD to potentially improve the interaction between ISM1 and ACVR1B ECD . CM samples were immunoprecipitated with anti-ISM1 antibodies and immunoblotted with anti-HA antibodies. ISM1 interaction with ACVR1B ECD is no longer observed when AMOP domain is absent. (E) Western blot of pSMAD2 in WCL of P19C6 cells treated with 100 ng/ml NODAL in the presence of mock, HIS-CER1, FLAG-LEFTY1, wild-type ISM1, ΔTSR1-ISM1, or ΔAMOP-ISM1 CM. Western blots for HIS (CER1), FLAG (LEFTY1), and ISM1 in CM samples is shown.
    Figure Legend Snippet: AMOP domain is required for the function of ISM1 as an antagonist of NODAL signaling. (A) Diagram of domain-deleted ISM1 constructs lacking either TSR1 or AMOP (ΔTSR1 and ΔAMOP, respectively). The N -glycosylation sites at positions N39 and N282 are indicated. N39 is located in the N terminus, 10 amino acids downstream from the signal peptide (amino acids 1–29), and N282 is located in the region between TSR1 and AMOP domains (amino acids 260–285). (B) Western blot of ISM1 in WCL and CM samples of HEK293T cells transiently transfected with wild-type ISM1, ΔTSR1-ISM1, or ΔAMOP-ISM1. Samples were digested with PNGase F or Endo H before being subjected to Western blotting. (C) Mapping of NODAL-interacting domain of ISM1. HEK293T cells were transiently transfected with FLAG-NODAL together with either wild-type ISM1 or domain-deleted ISM1 mutants. CM samples were immunoprecipitated (IP) with anti-FLAG or anti-ISM1 antibodies and subjected to Western blotting with anti-ISM1 and anti-FLAG antibodies. (D) Mapping of ACVR1B ECD -interacting domain of ISM1. HEK293T cells transiently expressing ACVR1B ECD were cotransfected with wild-type ISM1 or domain-deleted ISM1 mutants. ACVR2B ECD were also cotransfected with ACVR1B ECD to potentially improve the interaction between ISM1 and ACVR1B ECD . CM samples were immunoprecipitated with anti-ISM1 antibodies and immunoblotted with anti-HA antibodies. ISM1 interaction with ACVR1B ECD is no longer observed when AMOP domain is absent. (E) Western blot of pSMAD2 in WCL of P19C6 cells treated with 100 ng/ml NODAL in the presence of mock, HIS-CER1, FLAG-LEFTY1, wild-type ISM1, ΔTSR1-ISM1, or ΔAMOP-ISM1 CM. Western blots for HIS (CER1), FLAG (LEFTY1), and ISM1 in CM samples is shown.

    Techniques Used: Construct, Western Blot, Transfection, Immunoprecipitation, Expressing

    2) Product Images from "ISM1 regulates NODAL signaling and asymmetric organ morphogenesis during development"

    Article Title: ISM1 regulates NODAL signaling and asymmetric organ morphogenesis during development

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201801081

    The inhibitory effects of ISM1, LEFTY1, and CER1 on NODAL signaling. (A) Serum-starved HEK293T-CRIPTO cells treated with 100 ng/ml rNODAL in the presence of mock, LEFTY1, ISM1, or CER1 CM at indicated dilutions. WCL samples were subjected to Western blotting analyses for pSMAD2. Total SMAD2/3 and β-actin were used as internal controls. The protein levels of LEFTY1, ISM1, and CER1 representing their relative concentrations in the CMs were analyzed by Western blotting using Flag antibodies. (B) Quantification of the intensity of pSMAD2 relative to SMAD2/3 in three biological replicates. Data represent mean ± SEM. The unpaired, two-tailed t test was used for statistical analyses. Statistically significant P values are indicated. (C) Dual luciferase reporter assay to determine the SMAD2/FOXH1 transcriptional activity in HEK293T-CRIPTO cells treated by 100 ng/ml rNODAL for 24 h, together with CM of mock, FLAG-LEFTY1, FLAG-ISM1, or FLAG-CER1 at the indicated dilutions. Data represent mean ± SEM of four independent experiments. P values of statistical analyses using unpaired, two-tailed t test are indicated in each individual experimental group.
    Figure Legend Snippet: The inhibitory effects of ISM1, LEFTY1, and CER1 on NODAL signaling. (A) Serum-starved HEK293T-CRIPTO cells treated with 100 ng/ml rNODAL in the presence of mock, LEFTY1, ISM1, or CER1 CM at indicated dilutions. WCL samples were subjected to Western blotting analyses for pSMAD2. Total SMAD2/3 and β-actin were used as internal controls. The protein levels of LEFTY1, ISM1, and CER1 representing their relative concentrations in the CMs were analyzed by Western blotting using Flag antibodies. (B) Quantification of the intensity of pSMAD2 relative to SMAD2/3 in three biological replicates. Data represent mean ± SEM. The unpaired, two-tailed t test was used for statistical analyses. Statistically significant P values are indicated. (C) Dual luciferase reporter assay to determine the SMAD2/FOXH1 transcriptional activity in HEK293T-CRIPTO cells treated by 100 ng/ml rNODAL for 24 h, together with CM of mock, FLAG-LEFTY1, FLAG-ISM1, or FLAG-CER1 at the indicated dilutions. Data represent mean ± SEM of four independent experiments. P values of statistical analyses using unpaired, two-tailed t test are indicated in each individual experimental group.

    Techniques Used: Western Blot, Two Tailed Test, Luciferase, Reporter Assay, Activity Assay

    AMOP domain is required for the function of ISM1 as an antagonist of NODAL signaling. (A) Diagram of domain-deleted ISM1 constructs lacking either TSR1 or AMOP (ΔTSR1 and ΔAMOP, respectively). The N -glycosylation sites at positions N39 and N282 are indicated. N39 is located in the N terminus, 10 amino acids downstream from the signal peptide (amino acids 1–29), and N282 is located in the region between TSR1 and AMOP domains (amino acids 260–285). (B) Western blot of ISM1 in WCL and CM samples of HEK293T cells transiently transfected with wild-type ISM1, ΔTSR1-ISM1, or ΔAMOP-ISM1. Samples were digested with PNGase F or Endo H before being subjected to Western blotting. (C) Mapping of NODAL-interacting domain of ISM1. HEK293T cells were transiently transfected with FLAG-NODAL together with either wild-type ISM1 or domain-deleted ISM1 mutants. CM samples were immunoprecipitated (IP) with anti-FLAG or anti-ISM1 antibodies and subjected to Western blotting with anti-ISM1 and anti-FLAG antibodies. (D) Mapping of ACVR1B ECD -interacting domain of ISM1. HEK293T cells transiently expressing ACVR1B ECD were cotransfected with wild-type ISM1 or domain-deleted ISM1 mutants. ACVR2B ECD were also cotransfected with ACVR1B ECD to potentially improve the interaction between ISM1 and ACVR1B ECD . CM samples were immunoprecipitated with anti-ISM1 antibodies and immunoblotted with anti-HA antibodies. ISM1 interaction with ACVR1B ECD is no longer observed when AMOP domain is absent. (E) Western blot of pSMAD2 in WCL of P19C6 cells treated with 100 ng/ml NODAL in the presence of mock, HIS-CER1, FLAG-LEFTY1, wild-type ISM1, ΔTSR1-ISM1, or ΔAMOP-ISM1 CM. Western blots for HIS (CER1), FLAG (LEFTY1), and ISM1 in CM samples is shown.
    Figure Legend Snippet: AMOP domain is required for the function of ISM1 as an antagonist of NODAL signaling. (A) Diagram of domain-deleted ISM1 constructs lacking either TSR1 or AMOP (ΔTSR1 and ΔAMOP, respectively). The N -glycosylation sites at positions N39 and N282 are indicated. N39 is located in the N terminus, 10 amino acids downstream from the signal peptide (amino acids 1–29), and N282 is located in the region between TSR1 and AMOP domains (amino acids 260–285). (B) Western blot of ISM1 in WCL and CM samples of HEK293T cells transiently transfected with wild-type ISM1, ΔTSR1-ISM1, or ΔAMOP-ISM1. Samples were digested with PNGase F or Endo H before being subjected to Western blotting. (C) Mapping of NODAL-interacting domain of ISM1. HEK293T cells were transiently transfected with FLAG-NODAL together with either wild-type ISM1 or domain-deleted ISM1 mutants. CM samples were immunoprecipitated (IP) with anti-FLAG or anti-ISM1 antibodies and subjected to Western blotting with anti-ISM1 and anti-FLAG antibodies. (D) Mapping of ACVR1B ECD -interacting domain of ISM1. HEK293T cells transiently expressing ACVR1B ECD were cotransfected with wild-type ISM1 or domain-deleted ISM1 mutants. ACVR2B ECD were also cotransfected with ACVR1B ECD to potentially improve the interaction between ISM1 and ACVR1B ECD . CM samples were immunoprecipitated with anti-ISM1 antibodies and immunoblotted with anti-HA antibodies. ISM1 interaction with ACVR1B ECD is no longer observed when AMOP domain is absent. (E) Western blot of pSMAD2 in WCL of P19C6 cells treated with 100 ng/ml NODAL in the presence of mock, HIS-CER1, FLAG-LEFTY1, wild-type ISM1, ΔTSR1-ISM1, or ΔAMOP-ISM1 CM. Western blots for HIS (CER1), FLAG (LEFTY1), and ISM1 in CM samples is shown.

    Techniques Used: Construct, Western Blot, Transfection, Immunoprecipitation, Expressing

    3) Product Images from "ISM1 regulates NODAL signaling and asymmetric organ morphogenesis during development"

    Article Title: ISM1 regulates NODAL signaling and asymmetric organ morphogenesis during development

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201801081

    The inhibitory effects of ISM1, LEFTY1, and CER1 on NODAL signaling. (A) Serum-starved HEK293T-CRIPTO cells treated with 100 ng/ml rNODAL in the presence of mock, LEFTY1, ISM1, or CER1 CM at indicated dilutions. WCL samples were subjected to Western blotting analyses for pSMAD2. Total SMAD2/3 and β-actin were used as internal controls. The protein levels of LEFTY1, ISM1, and CER1 representing their relative concentrations in the CMs were analyzed by Western blotting using Flag antibodies. (B) Quantification of the intensity of pSMAD2 relative to SMAD2/3 in three biological replicates. Data represent mean ± SEM. The unpaired, two-tailed t test was used for statistical analyses. Statistically significant P values are indicated. (C) Dual luciferase reporter assay to determine the SMAD2/FOXH1 transcriptional activity in HEK293T-CRIPTO cells treated by 100 ng/ml rNODAL for 24 h, together with CM of mock, FLAG-LEFTY1, FLAG-ISM1, or FLAG-CER1 at the indicated dilutions. Data represent mean ± SEM of four independent experiments. P values of statistical analyses using unpaired, two-tailed t test are indicated in each individual experimental group.
    Figure Legend Snippet: The inhibitory effects of ISM1, LEFTY1, and CER1 on NODAL signaling. (A) Serum-starved HEK293T-CRIPTO cells treated with 100 ng/ml rNODAL in the presence of mock, LEFTY1, ISM1, or CER1 CM at indicated dilutions. WCL samples were subjected to Western blotting analyses for pSMAD2. Total SMAD2/3 and β-actin were used as internal controls. The protein levels of LEFTY1, ISM1, and CER1 representing their relative concentrations in the CMs were analyzed by Western blotting using Flag antibodies. (B) Quantification of the intensity of pSMAD2 relative to SMAD2/3 in three biological replicates. Data represent mean ± SEM. The unpaired, two-tailed t test was used for statistical analyses. Statistically significant P values are indicated. (C) Dual luciferase reporter assay to determine the SMAD2/FOXH1 transcriptional activity in HEK293T-CRIPTO cells treated by 100 ng/ml rNODAL for 24 h, together with CM of mock, FLAG-LEFTY1, FLAG-ISM1, or FLAG-CER1 at the indicated dilutions. Data represent mean ± SEM of four independent experiments. P values of statistical analyses using unpaired, two-tailed t test are indicated in each individual experimental group.

    Techniques Used: Western Blot, Two Tailed Test, Luciferase, Reporter Assay, Activity Assay

    AMOP domain is required for the function of ISM1 as an antagonist of NODAL signaling. (A) Diagram of domain-deleted ISM1 constructs lacking either TSR1 or AMOP (ΔTSR1 and ΔAMOP, respectively). The N -glycosylation sites at positions N39 and N282 are indicated. N39 is located in the N terminus, 10 amino acids downstream from the signal peptide (amino acids 1–29), and N282 is located in the region between TSR1 and AMOP domains (amino acids 260–285). (B) Western blot of ISM1 in WCL and CM samples of HEK293T cells transiently transfected with wild-type ISM1, ΔTSR1-ISM1, or ΔAMOP-ISM1. Samples were digested with PNGase F or Endo H before being subjected to Western blotting. (C) Mapping of NODAL-interacting domain of ISM1. HEK293T cells were transiently transfected with FLAG-NODAL together with either wild-type ISM1 or domain-deleted ISM1 mutants. CM samples were immunoprecipitated (IP) with anti-FLAG or anti-ISM1 antibodies and subjected to Western blotting with anti-ISM1 and anti-FLAG antibodies. (D) Mapping of ACVR1B ECD -interacting domain of ISM1. HEK293T cells transiently expressing ACVR1B ECD were cotransfected with wild-type ISM1 or domain-deleted ISM1 mutants. ACVR2B ECD were also cotransfected with ACVR1B ECD to potentially improve the interaction between ISM1 and ACVR1B ECD . CM samples were immunoprecipitated with anti-ISM1 antibodies and immunoblotted with anti-HA antibodies. ISM1 interaction with ACVR1B ECD is no longer observed when AMOP domain is absent. (E) Western blot of pSMAD2 in WCL of P19C6 cells treated with 100 ng/ml NODAL in the presence of mock, HIS-CER1, FLAG-LEFTY1, wild-type ISM1, ΔTSR1-ISM1, or ΔAMOP-ISM1 CM. Western blots for HIS (CER1), FLAG (LEFTY1), and ISM1 in CM samples is shown.
    Figure Legend Snippet: AMOP domain is required for the function of ISM1 as an antagonist of NODAL signaling. (A) Diagram of domain-deleted ISM1 constructs lacking either TSR1 or AMOP (ΔTSR1 and ΔAMOP, respectively). The N -glycosylation sites at positions N39 and N282 are indicated. N39 is located in the N terminus, 10 amino acids downstream from the signal peptide (amino acids 1–29), and N282 is located in the region between TSR1 and AMOP domains (amino acids 260–285). (B) Western blot of ISM1 in WCL and CM samples of HEK293T cells transiently transfected with wild-type ISM1, ΔTSR1-ISM1, or ΔAMOP-ISM1. Samples were digested with PNGase F or Endo H before being subjected to Western blotting. (C) Mapping of NODAL-interacting domain of ISM1. HEK293T cells were transiently transfected with FLAG-NODAL together with either wild-type ISM1 or domain-deleted ISM1 mutants. CM samples were immunoprecipitated (IP) with anti-FLAG or anti-ISM1 antibodies and subjected to Western blotting with anti-ISM1 and anti-FLAG antibodies. (D) Mapping of ACVR1B ECD -interacting domain of ISM1. HEK293T cells transiently expressing ACVR1B ECD were cotransfected with wild-type ISM1 or domain-deleted ISM1 mutants. ACVR2B ECD were also cotransfected with ACVR1B ECD to potentially improve the interaction between ISM1 and ACVR1B ECD . CM samples were immunoprecipitated with anti-ISM1 antibodies and immunoblotted with anti-HA antibodies. ISM1 interaction with ACVR1B ECD is no longer observed when AMOP domain is absent. (E) Western blot of pSMAD2 in WCL of P19C6 cells treated with 100 ng/ml NODAL in the presence of mock, HIS-CER1, FLAG-LEFTY1, wild-type ISM1, ΔTSR1-ISM1, or ΔAMOP-ISM1 CM. Western blots for HIS (CER1), FLAG (LEFTY1), and ISM1 in CM samples is shown.

    Techniques Used: Construct, Western Blot, Transfection, Immunoprecipitation, Expressing

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    Sino Biological cer1 flag plasmids
    The inhibitory effects of ISM1, LEFTY1, and <t>CER1</t> on NODAL signaling. (A) Serum-starved HEK293T-CRIPTO cells treated with 100 ng/ml rNODAL in the presence of mock, LEFTY1, ISM1, or CER1 CM at indicated dilutions. WCL samples were subjected to Western blotting analyses for pSMAD2. Total SMAD2/3 and β-actin were used as internal controls. The protein levels of LEFTY1, ISM1, and CER1 representing their relative concentrations in the CMs were analyzed by Western blotting using <t>Flag</t> antibodies. (B) Quantification of the intensity of pSMAD2 relative to SMAD2/3 in three biological replicates. Data represent mean ± SEM. The unpaired, two-tailed t test was used for statistical analyses. Statistically significant P values are indicated. (C) Dual luciferase reporter assay to determine the SMAD2/FOXH1 transcriptional activity in HEK293T-CRIPTO cells treated by 100 ng/ml rNODAL for 24 h, together with CM of mock, FLAG-LEFTY1, FLAG-ISM1, or FLAG-CER1 at the indicated dilutions. Data represent mean ± SEM of four independent experiments. P values of statistical analyses using unpaired, two-tailed t test are indicated in each individual experimental group.
    Cer1 Flag Plasmids, supplied by Sino Biological, 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/cer1 flag plasmids/product/Sino Biological
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cer1 flag plasmids - by Bioz Stars, 2021-05
    86/100 stars
      Buy from Supplier

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    The inhibitory effects of ISM1, LEFTY1, and CER1 on NODAL signaling. (A) Serum-starved HEK293T-CRIPTO cells treated with 100 ng/ml rNODAL in the presence of mock, LEFTY1, ISM1, or CER1 CM at indicated dilutions. WCL samples were subjected to Western blotting analyses for pSMAD2. Total SMAD2/3 and β-actin were used as internal controls. The protein levels of LEFTY1, ISM1, and CER1 representing their relative concentrations in the CMs were analyzed by Western blotting using Flag antibodies. (B) Quantification of the intensity of pSMAD2 relative to SMAD2/3 in three biological replicates. Data represent mean ± SEM. The unpaired, two-tailed t test was used for statistical analyses. Statistically significant P values are indicated. (C) Dual luciferase reporter assay to determine the SMAD2/FOXH1 transcriptional activity in HEK293T-CRIPTO cells treated by 100 ng/ml rNODAL for 24 h, together with CM of mock, FLAG-LEFTY1, FLAG-ISM1, or FLAG-CER1 at the indicated dilutions. Data represent mean ± SEM of four independent experiments. P values of statistical analyses using unpaired, two-tailed t test are indicated in each individual experimental group.

    Journal: The Journal of Cell Biology

    Article Title: ISM1 regulates NODAL signaling and asymmetric organ morphogenesis during development

    doi: 10.1083/jcb.201801081

    Figure Lengend Snippet: The inhibitory effects of ISM1, LEFTY1, and CER1 on NODAL signaling. (A) Serum-starved HEK293T-CRIPTO cells treated with 100 ng/ml rNODAL in the presence of mock, LEFTY1, ISM1, or CER1 CM at indicated dilutions. WCL samples were subjected to Western blotting analyses for pSMAD2. Total SMAD2/3 and β-actin were used as internal controls. The protein levels of LEFTY1, ISM1, and CER1 representing their relative concentrations in the CMs were analyzed by Western blotting using Flag antibodies. (B) Quantification of the intensity of pSMAD2 relative to SMAD2/3 in three biological replicates. Data represent mean ± SEM. The unpaired, two-tailed t test was used for statistical analyses. Statistically significant P values are indicated. (C) Dual luciferase reporter assay to determine the SMAD2/FOXH1 transcriptional activity in HEK293T-CRIPTO cells treated by 100 ng/ml rNODAL for 24 h, together with CM of mock, FLAG-LEFTY1, FLAG-ISM1, or FLAG-CER1 at the indicated dilutions. Data represent mean ± SEM of four independent experiments. P values of statistical analyses using unpaired, two-tailed t test are indicated in each individual experimental group.

    Article Snippet: MYC-ACVR2A and CER1-FLAG plasmids were purchased from Sino Biological (MG50613-NM and MG51161-CF, respectively).

    Techniques: Western Blot, Two Tailed Test, Luciferase, Reporter Assay, Activity Assay

    AMOP domain is required for the function of ISM1 as an antagonist of NODAL signaling. (A) Diagram of domain-deleted ISM1 constructs lacking either TSR1 or AMOP (ΔTSR1 and ΔAMOP, respectively). The N -glycosylation sites at positions N39 and N282 are indicated. N39 is located in the N terminus, 10 amino acids downstream from the signal peptide (amino acids 1–29), and N282 is located in the region between TSR1 and AMOP domains (amino acids 260–285). (B) Western blot of ISM1 in WCL and CM samples of HEK293T cells transiently transfected with wild-type ISM1, ΔTSR1-ISM1, or ΔAMOP-ISM1. Samples were digested with PNGase F or Endo H before being subjected to Western blotting. (C) Mapping of NODAL-interacting domain of ISM1. HEK293T cells were transiently transfected with FLAG-NODAL together with either wild-type ISM1 or domain-deleted ISM1 mutants. CM samples were immunoprecipitated (IP) with anti-FLAG or anti-ISM1 antibodies and subjected to Western blotting with anti-ISM1 and anti-FLAG antibodies. (D) Mapping of ACVR1B ECD -interacting domain of ISM1. HEK293T cells transiently expressing ACVR1B ECD were cotransfected with wild-type ISM1 or domain-deleted ISM1 mutants. ACVR2B ECD were also cotransfected with ACVR1B ECD to potentially improve the interaction between ISM1 and ACVR1B ECD . CM samples were immunoprecipitated with anti-ISM1 antibodies and immunoblotted with anti-HA antibodies. ISM1 interaction with ACVR1B ECD is no longer observed when AMOP domain is absent. (E) Western blot of pSMAD2 in WCL of P19C6 cells treated with 100 ng/ml NODAL in the presence of mock, HIS-CER1, FLAG-LEFTY1, wild-type ISM1, ΔTSR1-ISM1, or ΔAMOP-ISM1 CM. Western blots for HIS (CER1), FLAG (LEFTY1), and ISM1 in CM samples is shown.

    Journal: The Journal of Cell Biology

    Article Title: ISM1 regulates NODAL signaling and asymmetric organ morphogenesis during development

    doi: 10.1083/jcb.201801081

    Figure Lengend Snippet: AMOP domain is required for the function of ISM1 as an antagonist of NODAL signaling. (A) Diagram of domain-deleted ISM1 constructs lacking either TSR1 or AMOP (ΔTSR1 and ΔAMOP, respectively). The N -glycosylation sites at positions N39 and N282 are indicated. N39 is located in the N terminus, 10 amino acids downstream from the signal peptide (amino acids 1–29), and N282 is located in the region between TSR1 and AMOP domains (amino acids 260–285). (B) Western blot of ISM1 in WCL and CM samples of HEK293T cells transiently transfected with wild-type ISM1, ΔTSR1-ISM1, or ΔAMOP-ISM1. Samples were digested with PNGase F or Endo H before being subjected to Western blotting. (C) Mapping of NODAL-interacting domain of ISM1. HEK293T cells were transiently transfected with FLAG-NODAL together with either wild-type ISM1 or domain-deleted ISM1 mutants. CM samples were immunoprecipitated (IP) with anti-FLAG or anti-ISM1 antibodies and subjected to Western blotting with anti-ISM1 and anti-FLAG antibodies. (D) Mapping of ACVR1B ECD -interacting domain of ISM1. HEK293T cells transiently expressing ACVR1B ECD were cotransfected with wild-type ISM1 or domain-deleted ISM1 mutants. ACVR2B ECD were also cotransfected with ACVR1B ECD to potentially improve the interaction between ISM1 and ACVR1B ECD . CM samples were immunoprecipitated with anti-ISM1 antibodies and immunoblotted with anti-HA antibodies. ISM1 interaction with ACVR1B ECD is no longer observed when AMOP domain is absent. (E) Western blot of pSMAD2 in WCL of P19C6 cells treated with 100 ng/ml NODAL in the presence of mock, HIS-CER1, FLAG-LEFTY1, wild-type ISM1, ΔTSR1-ISM1, or ΔAMOP-ISM1 CM. Western blots for HIS (CER1), FLAG (LEFTY1), and ISM1 in CM samples is shown.

    Article Snippet: MYC-ACVR2A and CER1-FLAG plasmids were purchased from Sino Biological (MG50613-NM and MG51161-CF, respectively).

    Techniques: Construct, Western Blot, Transfection, Immunoprecipitation, Expressing

    The inhibitory effects of ISM1, LEFTY1, and CER1 on NODAL signaling. (A) Serum-starved HEK293T-CRIPTO cells treated with 100 ng/ml rNODAL in the presence of mock, LEFTY1, ISM1, or CER1 CM at indicated dilutions. WCL samples were subjected to Western blotting analyses for pSMAD2. Total SMAD2/3 and β-actin were used as internal controls. The protein levels of LEFTY1, ISM1, and CER1 representing their relative concentrations in the CMs were analyzed by Western blotting using Flag antibodies. (B) Quantification of the intensity of pSMAD2 relative to SMAD2/3 in three biological replicates. Data represent mean ± SEM. The unpaired, two-tailed t test was used for statistical analyses. Statistically significant P values are indicated. (C) Dual luciferase reporter assay to determine the SMAD2/FOXH1 transcriptional activity in HEK293T-CRIPTO cells treated by 100 ng/ml rNODAL for 24 h, together with CM of mock, FLAG-LEFTY1, FLAG-ISM1, or FLAG-CER1 at the indicated dilutions. Data represent mean ± SEM of four independent experiments. P values of statistical analyses using unpaired, two-tailed t test are indicated in each individual experimental group.

    Journal: The Journal of Cell Biology

    Article Title: ISM1 regulates NODAL signaling and asymmetric organ morphogenesis during development

    doi: 10.1083/jcb.201801081

    Figure Lengend Snippet: The inhibitory effects of ISM1, LEFTY1, and CER1 on NODAL signaling. (A) Serum-starved HEK293T-CRIPTO cells treated with 100 ng/ml rNODAL in the presence of mock, LEFTY1, ISM1, or CER1 CM at indicated dilutions. WCL samples were subjected to Western blotting analyses for pSMAD2. Total SMAD2/3 and β-actin were used as internal controls. The protein levels of LEFTY1, ISM1, and CER1 representing their relative concentrations in the CMs were analyzed by Western blotting using Flag antibodies. (B) Quantification of the intensity of pSMAD2 relative to SMAD2/3 in three biological replicates. Data represent mean ± SEM. The unpaired, two-tailed t test was used for statistical analyses. Statistically significant P values are indicated. (C) Dual luciferase reporter assay to determine the SMAD2/FOXH1 transcriptional activity in HEK293T-CRIPTO cells treated by 100 ng/ml rNODAL for 24 h, together with CM of mock, FLAG-LEFTY1, FLAG-ISM1, or FLAG-CER1 at the indicated dilutions. Data represent mean ± SEM of four independent experiments. P values of statistical analyses using unpaired, two-tailed t test are indicated in each individual experimental group.

    Article Snippet: MYC-ACVR2A and CER1-FLAG plasmids were purchased from Sino Biological (MG50613-NM and MG51161-CF, respectively).

    Techniques: Western Blot, Two Tailed Test, Luciferase, Reporter Assay, Activity Assay

    AMOP domain is required for the function of ISM1 as an antagonist of NODAL signaling. (A) Diagram of domain-deleted ISM1 constructs lacking either TSR1 or AMOP (ΔTSR1 and ΔAMOP, respectively). The N -glycosylation sites at positions N39 and N282 are indicated. N39 is located in the N terminus, 10 amino acids downstream from the signal peptide (amino acids 1–29), and N282 is located in the region between TSR1 and AMOP domains (amino acids 260–285). (B) Western blot of ISM1 in WCL and CM samples of HEK293T cells transiently transfected with wild-type ISM1, ΔTSR1-ISM1, or ΔAMOP-ISM1. Samples were digested with PNGase F or Endo H before being subjected to Western blotting. (C) Mapping of NODAL-interacting domain of ISM1. HEK293T cells were transiently transfected with FLAG-NODAL together with either wild-type ISM1 or domain-deleted ISM1 mutants. CM samples were immunoprecipitated (IP) with anti-FLAG or anti-ISM1 antibodies and subjected to Western blotting with anti-ISM1 and anti-FLAG antibodies. (D) Mapping of ACVR1B ECD -interacting domain of ISM1. HEK293T cells transiently expressing ACVR1B ECD were cotransfected with wild-type ISM1 or domain-deleted ISM1 mutants. ACVR2B ECD were also cotransfected with ACVR1B ECD to potentially improve the interaction between ISM1 and ACVR1B ECD . CM samples were immunoprecipitated with anti-ISM1 antibodies and immunoblotted with anti-HA antibodies. ISM1 interaction with ACVR1B ECD is no longer observed when AMOP domain is absent. (E) Western blot of pSMAD2 in WCL of P19C6 cells treated with 100 ng/ml NODAL in the presence of mock, HIS-CER1, FLAG-LEFTY1, wild-type ISM1, ΔTSR1-ISM1, or ΔAMOP-ISM1 CM. Western blots for HIS (CER1), FLAG (LEFTY1), and ISM1 in CM samples is shown.

    Journal: The Journal of Cell Biology

    Article Title: ISM1 regulates NODAL signaling and asymmetric organ morphogenesis during development

    doi: 10.1083/jcb.201801081

    Figure Lengend Snippet: AMOP domain is required for the function of ISM1 as an antagonist of NODAL signaling. (A) Diagram of domain-deleted ISM1 constructs lacking either TSR1 or AMOP (ΔTSR1 and ΔAMOP, respectively). The N -glycosylation sites at positions N39 and N282 are indicated. N39 is located in the N terminus, 10 amino acids downstream from the signal peptide (amino acids 1–29), and N282 is located in the region between TSR1 and AMOP domains (amino acids 260–285). (B) Western blot of ISM1 in WCL and CM samples of HEK293T cells transiently transfected with wild-type ISM1, ΔTSR1-ISM1, or ΔAMOP-ISM1. Samples were digested with PNGase F or Endo H before being subjected to Western blotting. (C) Mapping of NODAL-interacting domain of ISM1. HEK293T cells were transiently transfected with FLAG-NODAL together with either wild-type ISM1 or domain-deleted ISM1 mutants. CM samples were immunoprecipitated (IP) with anti-FLAG or anti-ISM1 antibodies and subjected to Western blotting with anti-ISM1 and anti-FLAG antibodies. (D) Mapping of ACVR1B ECD -interacting domain of ISM1. HEK293T cells transiently expressing ACVR1B ECD were cotransfected with wild-type ISM1 or domain-deleted ISM1 mutants. ACVR2B ECD were also cotransfected with ACVR1B ECD to potentially improve the interaction between ISM1 and ACVR1B ECD . CM samples were immunoprecipitated with anti-ISM1 antibodies and immunoblotted with anti-HA antibodies. ISM1 interaction with ACVR1B ECD is no longer observed when AMOP domain is absent. (E) Western blot of pSMAD2 in WCL of P19C6 cells treated with 100 ng/ml NODAL in the presence of mock, HIS-CER1, FLAG-LEFTY1, wild-type ISM1, ΔTSR1-ISM1, or ΔAMOP-ISM1 CM. Western blots for HIS (CER1), FLAG (LEFTY1), and ISM1 in CM samples is shown.

    Article Snippet: MYC-ACVR2A and CER1-FLAG plasmids were purchased from Sino Biological (MG50613-NM and MG51161-CF, respectively).

    Techniques: Construct, Western Blot, Transfection, Immunoprecipitation, Expressing