β trcp2  (Proteintech)

Journal: Science Advances

Article Title: WWC proteins–mediated compensatory mechanism restricts schwannomatosis driven by NF2 loss of function

doi: 10.1126/sciadv.adp4765

Figure Lengend Snippet: ( A ) WWC1-3 ubiquitination. HEK293A cells were cotransfected with FLAG-tagged WWC1-3 and hemagglutinin (HA)–tagged ubiquitin, immunoprecipitated WWC proteins were subjected to immunoblotting using HA antibody. Cells were treated with bortezomib (BTZ) for 6 hours before IP assay. ( B ) Schematic diagram of Bio-ID and mass spectrometry procedures. ( C ) Known interacting proteins and identified E3 ligases for WWC1. ( D ) Elevated protein levels of WWC1-3 in BTRC and BTRCP2 dKO HEK293A cells, with quantification shown. Asterisks indicate nonspecific bands. ( E ) Decreased WWC1-3 levels in HEK293A cells with ectopic β-TrCP1 or β-TrCP2. ( F ) WWC1-3 interact with β-TrCP1/2. HA-tagged WWC1/2/3 were cotransfected with FLAG-tagged β-TrCP1/2 into HEK293A cells, respectively, and subjected to coimmunoprecipitation (co-IP) assay. ( G ) Increased ubiquitination of WWC1 in cells with ectopic expression of β-TrCP1 and/or β-TrCP2. MYC-β-TrCP1 and β-TrCP2 were cotransfected with FLAG-WWC1 and HA-ubiquitin (Ub). WWC1 was immunoprecipitated, and ubiquitination was detected by HA antibody. Cells were treated with BTZ for 6 hours before IP assay. ( H ) Amino acids 601 to 700 of WWC1 are essential for interaction with β-TrCP1. WT or truncated WWC1 was cotransfected with HA-tagged β-TrCP1 and subjected to co-IP assay. ( I ) S631 is essential for WWC1 to interact with β-TrCP1/2. WT or S631A FLAG-WWC1 was cotransfected with HA-β-TrCP2 or MYC-β-TrCP1 and subjected to co-IP assay. ( J ) S631A mutant WWC1 shows impaired ubiquitination. WT or S631A FLAG-WWC1 was cotransfected with indicated plasmids into HEK293A cells, WWC1 was immunoprecipitated, and ubiquitination was determined. Cells were treated with BTZ for 6 hours before IP assay. ( K ) S631A mutant WWC1 is more stable. WT or S631A WWC1 was overexpressed in HEK293A cells, treated with CHX (100 μg/ml) for 4 to 12 hours, and whole-cell lysate was collected for immunoblotting, and protein levels of WWC1 were quantified.

Article Snippet: Primary antibodies specific to WWC1 and β-TrCP2 (13149-1-AP, Proteintech) were used for detecting WWC1 and β-TrCP2 interaction.

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Mass Spectrometry, Co-Immunoprecipitation Assay, Expressing, Mutagenesis

Journal: Science Advances

Article Title: WWC proteins–mediated compensatory mechanism restricts schwannomatosis driven by NF2 loss of function

doi: 10.1126/sciadv.adp4765

Figure Lengend Snippet: ( A ) Reduced ubiquitination of WWC1/2 in NF2 KO cells. WT and NF2 KO HEK293A cells were cotransfected with FLAG-WWC1/2 and HA-Ub, WWC1/2 were immunoprecipitated, and ubiquitination were determined by immunoblotting. Cells were treated with BTZ for 6 hours before IP assay. ( B and C ) Concurrent deletion of β-TrCP1 and β-TrCP2 induces expression of WWC1-3 in WT but not NF2 KO cells. Two sets of small guide RNAs targeting BTRC and BTRCP2 and Cas9 plasmids were stably expressed in WT or NF2 KO HEK293A cells, and whole-cell lysate was collected for immunoblotting. WWC1-3 protein levels were also quantified. The asterisk indicates nonspecific bands. ( D ) NF2 interacts with β-TrCP1 and β-TrCP2. FLAG–β-TrCP1 and β-TrCP2 were expressed in HEK293A cells and immunoprecipitated, and co-immunoprecipitated endogenous NF2 was determined by immunoblotting. ( E ) NF2 mediates interaction between WWC1 and β-TrCP1/2. WT and NF2 KO HEK293A cells were cotransfected with indicated plasmids, WWC1 was immunoprecipitated, and bound β-TrCP1/2 was analyzed by immunoblotting and quantified. ( F ) Reduced interaction between WWC1 and β-TrCP2 in NF2 KO MDCK cells. PLA was used to detect the interaction between endogenous WWC1 and β-TrCP2. Maximum intensity projection from z-stack confocal images was used to show PLA spots (red); the nucleus was stained with DAPI (4′,6-diamidino-2-phenylindole). At least 200 cells from 20 microscopic fields per sample were used for quantification. ( G ) S631 is required for the regulation of WWC1 stability by NF2. WT, S631A, or WW domain–deleted (WW del) WWC1 was stably expressed with NF2 in NF2 ; WWC1-3 4KO HEK293A cells. The protein levels of WWC1 were quantified. ( H ) A schematic diagram shows the working mechanism underlying NF2-mediated regulation of WWC1-3 by recruiting β-TrCP1/2. NF2 acts as a platform to recruit WWC1-3 and β-TrCP1/2 together and promote WWC1-3 ubiquitination and degradation through proteasome.

Article Snippet: Primary antibodies specific to WWC1 and β-TrCP2 (13149-1-AP, Proteintech) were used for detecting WWC1 and β-TrCP2 interaction.

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Expressing, Stable Transfection, Staining

Journal: Science Advances

Article Title: WWC proteins–mediated compensatory mechanism restricts schwannomatosis driven by NF2 loss of function

doi: 10.1126/sciadv.adp4765

Figure Lengend Snippet: ( A ) The domain organization of WWC family proteins. WWC family contains three members: WWC1, WWC2, and WWC3. WW domain, coiled coil domain, C2 domain, and PDZ-binding motif are highly conserved in WWC family members. ( B ) The expression of Hippo pathway components in NF2 KO cells. WWC1-3 was robustly induced in HEK293A cells deficient in NF2. ( C ) Elevated expression of WWC1-3 in NF2 KO cells. Among HEK293A cells deficient with different Hippo pathway genes, the expression of WWC1-3 was only consistently induced in NF2 KO cells. Protein quantification is also shown. ( D ) Elevated expression of WWC1-3 in NF2 KO cells. Two different KO HEK293A cell lines were used. Protein quantification is also shown. ( E ) Restoration of WWC1-3 expression by ectopic NF2 in NF2 KO cells. Overexpression of NF2 had no robust effect on the WWC protein levels in WT HEK293A cells but reduced expression of WWC proteins in NF2 KO HEK293A cells. ( F ) Reduced WWC1/2 protein levels in HEI-193 cells upon NF2 expression. Protein quantification is shown. ( G ) Stabilization of WWC1-3 in NF2 KO cells. WT and NF2 KO HEK293A cells were treated with protein synthesis inhibitor CHX (100 μg/ml) for 2 to 12 hours. WWC1-3 protein levels were determined and quantified.

Article Snippet: Primary antibodies specific to WWC1 and β-TrCP2 (13149-1-AP, Proteintech) were used for detecting WWC1 and β-TrCP2 interaction.

Techniques: Binding Assay, Expressing, Over Expression

Journal: Science Advances

Article Title: WWC proteins–mediated compensatory mechanism restricts schwannomatosis driven by NF2 loss of function

doi: 10.1126/sciadv.adp4765

Figure Lengend Snippet: ( A ) WWC1-3 ubiquitination. HEK293A cells were cotransfected with FLAG-tagged WWC1-3 and hemagglutinin (HA)–tagged ubiquitin, immunoprecipitated WWC proteins were subjected to immunoblotting using HA antibody. Cells were treated with bortezomib (BTZ) for 6 hours before IP assay. ( B ) Schematic diagram of Bio-ID and mass spectrometry procedures. ( C ) Known interacting proteins and identified E3 ligases for WWC1. ( D ) Elevated protein levels of WWC1-3 in BTRC and BTRCP2 dKO HEK293A cells, with quantification shown. Asterisks indicate nonspecific bands. ( E ) Decreased WWC1-3 levels in HEK293A cells with ectopic β-TrCP1 or β-TrCP2. ( F ) WWC1-3 interact with β-TrCP1/2. HA-tagged WWC1/2/3 were cotransfected with FLAG-tagged β-TrCP1/2 into HEK293A cells, respectively, and subjected to coimmunoprecipitation (co-IP) assay. ( G ) Increased ubiquitination of WWC1 in cells with ectopic expression of β-TrCP1 and/or β-TrCP2. MYC-β-TrCP1 and β-TrCP2 were cotransfected with FLAG-WWC1 and HA-ubiquitin (Ub). WWC1 was immunoprecipitated, and ubiquitination was detected by HA antibody. Cells were treated with BTZ for 6 hours before IP assay. ( H ) Amino acids 601 to 700 of WWC1 are essential for interaction with β-TrCP1. WT or truncated WWC1 was cotransfected with HA-tagged β-TrCP1 and subjected to co-IP assay. ( I ) S631 is essential for WWC1 to interact with β-TrCP1/2. WT or S631A FLAG-WWC1 was cotransfected with HA-β-TrCP2 or MYC-β-TrCP1 and subjected to co-IP assay. ( J ) S631A mutant WWC1 shows impaired ubiquitination. WT or S631A FLAG-WWC1 was cotransfected with indicated plasmids into HEK293A cells, WWC1 was immunoprecipitated, and ubiquitination was determined. Cells were treated with BTZ for 6 hours before IP assay. ( K ) S631A mutant WWC1 is more stable. WT or S631A WWC1 was overexpressed in HEK293A cells, treated with CHX (100 μg/ml) for 4 to 12 hours, and whole-cell lysate was collected for immunoblotting, and protein levels of WWC1 were quantified.

Article Snippet: Primary antibodies specific to WWC1 and β-TrCP2 (13149-1-AP, Proteintech) were used for detecting WWC1 and β-TrCP2 interaction.

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Mass Spectrometry, Co-Immunoprecipitation Assay, Expressing, Mutagenesis

Journal: Science Advances

Article Title: WWC proteins–mediated compensatory mechanism restricts schwannomatosis driven by NF2 loss of function

doi: 10.1126/sciadv.adp4765

Figure Lengend Snippet: ( A ) Reduced ubiquitination of WWC1/2 in NF2 KO cells. WT and NF2 KO HEK293A cells were cotransfected with FLAG-WWC1/2 and HA-Ub, WWC1/2 were immunoprecipitated, and ubiquitination were determined by immunoblotting. Cells were treated with BTZ for 6 hours before IP assay. ( B and C ) Concurrent deletion of β-TrCP1 and β-TrCP2 induces expression of WWC1-3 in WT but not NF2 KO cells. Two sets of small guide RNAs targeting BTRC and BTRCP2 and Cas9 plasmids were stably expressed in WT or NF2 KO HEK293A cells, and whole-cell lysate was collected for immunoblotting. WWC1-3 protein levels were also quantified. The asterisk indicates nonspecific bands. ( D ) NF2 interacts with β-TrCP1 and β-TrCP2. FLAG–β-TrCP1 and β-TrCP2 were expressed in HEK293A cells and immunoprecipitated, and co-immunoprecipitated endogenous NF2 was determined by immunoblotting. ( E ) NF2 mediates interaction between WWC1 and β-TrCP1/2. WT and NF2 KO HEK293A cells were cotransfected with indicated plasmids, WWC1 was immunoprecipitated, and bound β-TrCP1/2 was analyzed by immunoblotting and quantified. ( F ) Reduced interaction between WWC1 and β-TrCP2 in NF2 KO MDCK cells. PLA was used to detect the interaction between endogenous WWC1 and β-TrCP2. Maximum intensity projection from z-stack confocal images was used to show PLA spots (red); the nucleus was stained with DAPI (4′,6-diamidino-2-phenylindole). At least 200 cells from 20 microscopic fields per sample were used for quantification. ( G ) S631 is required for the regulation of WWC1 stability by NF2. WT, S631A, or WW domain–deleted (WW del) WWC1 was stably expressed with NF2 in NF2 ; WWC1-3 4KO HEK293A cells. The protein levels of WWC1 were quantified. ( H ) A schematic diagram shows the working mechanism underlying NF2-mediated regulation of WWC1-3 by recruiting β-TrCP1/2. NF2 acts as a platform to recruit WWC1-3 and β-TrCP1/2 together and promote WWC1-3 ubiquitination and degradation through proteasome.

Article Snippet: Primary antibodies specific to WWC1 and β-TrCP2 (13149-1-AP, Proteintech) were used for detecting WWC1 and β-TrCP2 interaction.

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Expressing, Stable Transfection, Staining

Journal: Science Advances

Article Title: WWC proteins–mediated compensatory mechanism restricts schwannomatosis driven by NF2 loss of function

doi: 10.1126/sciadv.adp4765

Figure Lengend Snippet: ( A ) A diagram illustrating the effects of WWC1-3 and NF2, as organizers of HPO1 and HPO2 signaling modules, respectively, on YAP/TAZ activity and tumorigenesis. ( B to E ) Concurrent deletion of NF2 and WWC1-3 results in YAP/TAZ hyperactivation. NF2 and/or WWC1-3 were knocked out in HEK293A [(B) and (C)] and HEI-193 [(D) and (E)] cells, expression of proteins [(B) and (D)] and YAP/TAZ target genes [(C) and (E)] were determined by immunoblotting and quantitative PCR, respectively. Active YAP is non-phospho-YAP. Pooled HEI-193 cells (indicated by knockdown, KD) were used in both biochemical and functional assays (D to J). ( F ) Robustly increased colony forming capability of HEI-193 cells deficient in both NF2 and WWC1-3 in two-dimensional (2D) culture. Pictures and quantification of colony formation assay were shown. ( G ) Robustly increased anchorage-independent growth of HEI-193 cells deficient in both NF2 and WWC1-3 in 3D culture. Pictures and quantification of soft agar assay were shown. ( H to J ) HEI-193 cells deficient in both NF2 and WWC1-3 effectively develop subcutaneous tumors in nude mice. Gross images (H), growth curves (I), and weight (J) of tumors were shown. Four mice (eight injection sites) per group were used.

Article Snippet: Primary antibodies specific to WWC1 and β-TrCP2 (13149-1-AP, Proteintech) were used for detecting WWC1 and β-TrCP2 interaction.

Techniques: Activity Assay, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Knockdown, Functional Assay, Colony Assay, Soft Agar Assay, Injection