fopflash constructs  (Addgene inc)

Journal: Scientific Reports

Article Title: Smurf1 promotes gastric cancer progression by regulating Axin2-dependent Wnt signaling pathway

doi: 10.1038/s41598-025-23707-3

Figure Lengend Snippet: Smurf1 promoted Wnt/β-catenin signaling activation. ( A ) Parental and Smurf1-overexpressing AGS cells were transfected with TOPflash and FOPflash reporters and treated with Wnt3a, respectively. Wnt/β-catenin signaling activation was assessed using the TOPflash/FOPflash dual luciferase reporter system. Relative luciferase units (RLU) were calculated to assess β-catenin-triggered transcription. After Smurf1 overexpression in AGS cells, cytoplasmic ( B and D ) and nuclear ( C and D ) β-catenin protein levels were measured using the western blot assay. β-actin and Lamin B1 served as cytoplasmic and nuclear markers, respectively. ( E ) After Smurf1 overexpression in AGS cells, β-catenin nuclear translocation was assessed using IF analysis. Red fluorescence indicated β-catenin, and blue fluorescence indicated the nucleus. The results were shown as the median (1st quartile and 3rd quartile). * p < 0.05, ** p < 0.01.

Article Snippet: After Smurf1 overexpression, AGS cells were co-transfected with TOPflash construct (80 ng, Merck) or FOPflash construct (Merck) and pRL-TK plasmid (8 ng, Promega, WI, USA) using PEI max transfection reagent.

Techniques: Activation Assay, Transfection, Luciferase, Over Expression, Western Blot, Translocation Assay, Fluorescence

Journal: American Journal of Cancer Research

Article Title: Ubiquitin-specific peptidase 2a (USP2a) deubiquitinates and stabilizes β-catenin

doi:

Figure Lengend Snippet: USP2a upregulates β-catenin protein and promotes its transcriptional activity. A. Each SFB-tagged deubiquitinase was co-transfected with MYC-tagged β-catenin into HEK293T cells, followed by pulldown with S-protein beads and immunoblotting with antibodies against FLAG and MYC. B. Either β-catenin-responsive TOPflash or its mutant FOPflash construct was co-transfected with each SFB-tagged deubiquitinase and Renilla luciferase into HEK293T cells. A dual luciferase assay was used to determine β-catenin activity. Firefly luciferase activity was normalized to Renilla luciferase activity. MYC-tagged β-catenin serves as a positive control. Error bars are S.D. C. SFB-tagged GFP, USP2a, USP26, and USP42 were co-transfected with MYC-tagged β-catenin into HEK293T cells, and pulled down with S-protein beads. Antibodies against MYC and FLAG were used to detect β-catenin and DUBs. CypB (cyclophilin B) serves as the loading control. D. Left panel: luciferase reporter assay validating that the 4 candidate deubiquitinases (USP2a, USP36, DUB3, and OTUD7B) promote the transcriptional activity of β-catenin. Error bars are S.D. ***: P < 0.001. Right panel: immunoblotting of HSP90 and SFB-tagged GFP, USP2a, USP36, DUB3, and OTUD7B in HEK293T cells. HSP90 serves as the loading control. T: TOPflash; F: FOPflash. E. Each SFB-tagged candidate deubiquitinase was co-transfected with HA-tagged ubiquitin and MYC-tagged β-catenin into HEK293T cells. After MG132 treatment for 6 hours, β-catenin was immunoprecipitated with a MYC-specific antibody, followed by immunoblotting with antibodies against HA and MYC. F. Immunoblotting of β-catenin, FLAG, and HSP90 in HEK293T cells transfected with SFB-tagged GFP, USP2a, USP36, DUB3, or OTUD7B. G. qPCR of CTNNB1 (the gene that encodes β-catenin) in HEK293T cells transfected with the empty vector, wild-type USP2a, or the catalytically inactive mutant (C276A). Error bars are S.D. n.s.: not significant. H. Immunoblotting of USP2a, β-catenin, and CypB in BT549 cells transduced with USP2 shRNAs. I. Immunoblotting of USP2a, β-catenin, and CypB in BT549 cells transduced with USP2 shRNA with or without ectopic expression of USP2a.

Article Snippet: HA-ubiquitin, TOPflash, and FOPflash constructs were from Addgene (plasmid number: 17608, 12456, and 12457).

Techniques: Activity Assay, Transfection, Western Blot, Mutagenesis, Construct, Luciferase, Positive Control, Control, Reporter Assay, Ubiquitin Proteomics, Immunoprecipitation, Plasmid Preparation, Transduction, shRNA, Expressing

Journal: American Journal of Cancer Research

Article Title: Ubiquitin-specific peptidase 2a (USP2a) deubiquitinates and stabilizes β-catenin

doi:

Figure Lengend Snippet: USP2a activates Wnt/β-catenin signaling. A. Immunoblotting of FLAG-USP2a, β-catenin, HSP90 (cytoplasmic marker), and Lamin B1 (nuclear marker) in cytoplasmic and nuclear fractions of HEK293T cells transfected with the empty vector or SFB-USP2a. B. Top panel: either β-catenin-responsive TOPflash or its mutant FOPflash construct was co-transfected with SFB-tagged GFP, USP2a, or USP2aC276A and Renilla luciferase into HEK293T cells. A dual luciferase assay was used to determine β-catenin activity. Firefly luciferase activity was normalized to Renilla luciferase activity. Error bars are S.D. **: P < 0.01. Bottom panel: immunoblotting of SFB-USP2a, SFB-USP2aC276A, and CypB in HEK293T cells. T: TOPflash; F: FOPflash. C. Top panel: either β-catenin-responsive TOPflash or its mutant FOPflash construct was co-transfected with the SFB vector or SFB-USP2a and Renilla luciferase into HEK293T cells. Cells were treated with DMSO or 3 µM ML364 for 24 hours. A dual luciferase assay was used to determine β-catenin activity. Firefly luciferase activity was normalized to Renilla luciferase activity, and then TOPflash reads were normalized to FOPflash reads. Error bars are S.E.M. *: P < 0.05; **: P < 0.01. Bottom panel: immunoblotting of SFB-USP2a and CypB in HEK293T cells. D: DMSO; M: ML364. D. Human Wnt signaling targets PCR array analysis of HEK293T cells transfected with SFB-USP2a. Gene expression levels in SFB-USP2a-transfected cells were compared to those in empty vector-transfected cells (log2 scale). E. qPCR of BIRC5, CUBN, FGF7, CDON, BTRC, ANTXR1, and IGF2 in HEK293T cells transfected with the empty vector, SFB-USP2a, or SFB-USP2aC276A. Error bars are S.E.M. *: P < 0.05; **: P < 0.01; ***: P < 0.001. F. qPCR of BIRC5, CUBN, FGF7, CDON, BTRC, ANTXR1, and IGF2 in BT549 cells transduced with USP2 shRNAs or a scramble control (Scr). Error bars are S.E.M. *: P < 0.05; **: P < 0.01; ***: P < 0.001; #: not detectable.

Article Snippet: HA-ubiquitin, TOPflash, and FOPflash constructs were from Addgene (plasmid number: 17608, 12456, and 12457).

Techniques: Western Blot, Marker, Transfection, Plasmid Preparation, Mutagenesis, Construct, Luciferase, Activity Assay, Gene Expression, Transduction, Control