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Addgene inc jeff wrana
Jeff Wrana, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/pcmv5+smad7+ha/pCMV5-Smad7-HA+(Plasmid+%2311733)/pm41760544-200-225-229
Average 93 stars, based on 20 article reviews

jeff wrana - by Bioz Stars, 2026-09

93/100 stars

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Polymerase Chain Reaction:

Article Title: RNF11 sequestration of the E3 ligase SMURF2 on membranes antagonizes SMAD7 down-regulation of transforming growth factor β signaling
Article Snippet: FLAG-SMURF1, FLAG-SMURF2, and FLAG-SMURF2 C716A were purchased from ADDGENE (plasmids 11752, 11746, and 11747). .. pFastBac1 hRnf11 (untagged) was made by PCR amplification of the RNF11 open reading frame from pcDNA3.1 hRnf11 using the primers hRnf11BamHIF and hRnf11XhoIR and subcloned into pFastBac1. pFASTBACHTA hSMAD7-HA was made by PCR amplification of the hSMAD7-HA open reading frame from pCMV5-SMAD7-HA (Addgene 11733) using the primers EcoRI SMAD7F and SalI SMAD7-HA R. pFASTBAC HTA FLAG-SMURF2 and FLAG-SMURF2C716A were made by PCR amplification of the open reading frame from the mammalian expression vectors FLAG-SMURF2 and C716A using the primers EcoRI SMURF2F and NotI SMURF2R. ..

Article Title: RNF11 sequestration of the E3 ligase SMURF2 on membranes antagonizes SMAD7 down-regulation of transforming growth factor β signaling
Article Snippet: FLAG-SMURF1, FLAG-SMURF2, and FLAG-SMURF2 C716A were purchased from ADDGENE (plasmids 11752, 11746, and 11747). .. Baculovirus expression vectors pFastBac1 hRnf11 (untagged) was made by PCR amplification of the RNF11 open reading frame from pcDNA3.1 hRnf11 using the primers hRnf11BamHIF and hRnf11XhoIR and subcloned into pFastBac1. pFASTBACHTA hSMAD7-HA was made by PCR amplification of the hSMAD7-HA open reading frame from pCMV5-SMAD7-HA (Addgene 11733) using the primers EcoRI SMAD7F and SalI SMAD7-HA R. pFASTBAC HTA FLAG-SMURF2 and FLAG-SMURF2C716A were made by PCR amplification of the open reading frame from the mammalian expression vectors FLAG-SMURF2 and C716A using the primers EcoRI SMURF2F and NotI SMURF2R. ..

Article Title: RNF11 sequestration of the E3 ligase SMURF2 on membranes antagonizes SMAD7 down-regulation of transforming growth factor β signaling
Article Snippet: FLAG-Smurf1, FLAG-Smurf2, FLAG-Smurf2 C716A were purchased from ADDGENE (plasmids 11752, 11746, 11747). .. Baculovirus expression vectors: pFastBac1 hRnf11 (untagged) was made by PCR amplification of the Rnf11 open reading frame from pcDNA3.1 hRnf11 using the primers hRnf11BamH1F and hRnf11 Xho1R and subcloned into pFastBac1. pFASTBACHTA hSmad7-HA was made by PCR amplification of the hSmad7-HA open reading frame from pCMV5-Smad7-HA (Addgene 11733) using the primers EcoR1 Smad7F and Sal1 Smad7-HA R. pFASTBAC HTA FLAG Smurf2 and FLAG Smurf2C716A were made by PCR amplification of the open reading frame from the mammalian expression vectors FLAG Smurf2 and C716A using the primers EcoR1 Smurf2F and Not1 Smurf2R. at H A C E T T E PE U N IV E R SIT Y on M arch 23, 2017 http://w w w .jbc.org/ D ow nloaded from Ring finger protein 11 sequesters Smurf2 on membranes 12 Antibodies and Affinity Resins All antibodies used in this study with the exception of the Rnf11 antibody were purchased from commercial sources. ..

Amplification:

Article Title: RNF11 sequestration of the E3 ligase SMURF2 on membranes antagonizes SMAD7 down-regulation of transforming growth factor β signaling
Article Snippet: FLAG-SMURF1, FLAG-SMURF2, and FLAG-SMURF2 C716A were purchased from ADDGENE (plasmids 11752, 11746, and 11747). .. Baculovirus expression vectors pFastBac1 hRnf11 (untagged) was made by PCR amplification of the RNF11 open reading frame from pcDNA3.1 hRnf11 using the primers hRnf11BamHIF and hRnf11XhoIR and subcloned into pFastBac1. pFASTBACHTA hSMAD7-HA was made by PCR amplification of the hSMAD7-HA open reading frame from pCMV5-SMAD7-HA (Addgene 11733) using the primers EcoRI SMAD7F and SalI SMAD7-HA R. pFASTBAC HTA FLAG-SMURF2 and FLAG-SMURF2C716A were made by PCR amplification of the open reading frame from the mammalian expression vectors FLAG-SMURF2 and C716A using the primers EcoRI SMURF2F and NotI SMURF2R. ..

Article Title: RNF11 sequestration of the E3 ligase SMURF2 on membranes antagonizes SMAD7 down-regulation of transforming growth factor β signaling
Article Snippet: FLAG-Smurf1, FLAG-Smurf2, FLAG-Smurf2 C716A were purchased from ADDGENE (plasmids 11752, 11746, 11747). .. Baculovirus expression vectors: pFastBac1 hRnf11 (untagged) was made by PCR amplification of the Rnf11 open reading frame from pcDNA3.1 hRnf11 using the primers hRnf11BamH1F and hRnf11 Xho1R and subcloned into pFastBac1. pFASTBACHTA hSmad7-HA was made by PCR amplification of the hSmad7-HA open reading frame from pCMV5-Smad7-HA (Addgene 11733) using the primers EcoR1 Smad7F and Sal1 Smad7-HA R. pFASTBAC HTA FLAG Smurf2 and FLAG Smurf2C716A were made by PCR amplification of the open reading frame from the mammalian expression vectors FLAG Smurf2 and C716A using the primers EcoR1 Smurf2F and Not1 Smurf2R. at H A C E T T E PE U N IV E R SIT Y on M arch 23, 2017 http://w w w .jbc.org/ D ow nloaded from Ring finger protein 11 sequesters Smurf2 on membranes 12 Antibodies and Affinity Resins All antibodies used in this study with the exception of the Rnf11 antibody were purchased from commercial sources. ..

Expressing:

Article Title: RNF11 sequestration of the E3 ligase SMURF2 on membranes antagonizes SMAD7 down-regulation of transforming growth factor β signaling
Article Snippet: FLAG-SMURF1, FLAG-SMURF2, and FLAG-SMURF2 C716A were purchased from ADDGENE (plasmids 11752, 11746, and 11747). .. Baculovirus expression vectors pFastBac1 hRnf11 (untagged) was made by PCR amplification of the RNF11 open reading frame from pcDNA3.1 hRnf11 using the primers hRnf11BamHIF and hRnf11XhoIR and subcloned into pFastBac1. pFASTBACHTA hSMAD7-HA was made by PCR amplification of the hSMAD7-HA open reading frame from pCMV5-SMAD7-HA (Addgene 11733) using the primers EcoRI SMAD7F and SalI SMAD7-HA R. pFASTBAC HTA FLAG-SMURF2 and FLAG-SMURF2C716A were made by PCR amplification of the open reading frame from the mammalian expression vectors FLAG-SMURF2 and C716A using the primers EcoRI SMURF2F and NotI SMURF2R. ..

Article Title: RNF11 sequestration of the E3 ligase SMURF2 on membranes antagonizes SMAD7 down-regulation of transforming growth factor β signaling
Article Snippet: FLAG-Smurf1, FLAG-Smurf2, FLAG-Smurf2 C716A were purchased from ADDGENE (plasmids 11752, 11746, 11747). .. Baculovirus expression vectors: pFastBac1 hRnf11 (untagged) was made by PCR amplification of the Rnf11 open reading frame from pcDNA3.1 hRnf11 using the primers hRnf11BamH1F and hRnf11 Xho1R and subcloned into pFastBac1. pFASTBACHTA hSmad7-HA was made by PCR amplification of the hSmad7-HA open reading frame from pCMV5-Smad7-HA (Addgene 11733) using the primers EcoR1 Smad7F and Sal1 Smad7-HA R. pFASTBAC HTA FLAG Smurf2 and FLAG Smurf2C716A were made by PCR amplification of the open reading frame from the mammalian expression vectors FLAG Smurf2 and C716A using the primers EcoR1 Smurf2F and Not1 Smurf2R. at H A C E T T E PE U N IV E R SIT Y on M arch 23, 2017 http://w w w .jbc.org/ D ow nloaded from Ring finger protein 11 sequesters Smurf2 on membranes 12 Antibodies and Affinity Resins All antibodies used in this study with the exception of the Rnf11 antibody were purchased from commercial sources. ..

Plasmid Preparation:

Article Title: Tissue-specific I-Smad mechanisms revealed by structure–function analysis in Drosophila
Article Snippet: pUAST-attB-Dad , PCR product from LD47465 with XhoI/XbaI sites added cloned into pUAST-attB , , Transformation vector for UAS-Dad. .. PCMV5-Smad7-HA , N/A , pCMV5-Smad7-HA was a gift from Jeff Wrana (plasmid #11733; https://n2t.net/addgene:11733 ; RRID:Addgene_11733; Addgene) ( ) , Source for Smad7L CDS. .. PCMV5-Smad7 , Deleted HA tag from pCMV5-Smad7-HA with PCR , Current study , .

Image Search Results

Journal: eLife

Article Title: Transcriptional regulation of cyclophilin D by BMP/Smad signaling and its role in osteogenic differentiation

doi: 10.7554/eLife.75023

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pCMV5-Smad7 (plasmid) , Addgene , #11733 , .

Techniques: Transfection, Construct, Plasmid Preparation, In Vivo, Isolation, Recombinant, Gene Knock-In, Sequencing, PCR Cloning, Chromatin Immunoprecipitation, Magnetic Beads, Luciferase, Reporter Assay

Isolated primary Flnb −/− AF cells exhibit increased TGFβ, ERK, and p38 signaling activation. a Western blots of protein lysates isolated from unstimulated primary AF cells (left column) and stimulated with 5 ng·mL −1 TGFβ-1 ligand for 30 min (right column) probed with antibodies against p-Smad 3, p-ERK1/2, and GAPDH. b Quantification of TGFβ-1-stimulated Western blot normalized against GAPDH levels showing a significant increase in Smad 3 phosphorylation in both unstimulated and stimulated primary AF cells. c Quantification of TGFβ-1-stimulated Western blot normalized against GAPDH levels showing no significant increase in ERK1/2 phosphorylation in either unstimulated or stimulated primary AF cells. d Western blots of isolated unstimulated primary AF cells (left column) and stimulated with 5 ng·mL −1 BMP-2 ligand for 30 min (right column) probed with antibodies against p-Smad 1/5/8, p-p38, and GAPDH. e Quantification of BMP-2-stimulated Western blot normalized against GAPDH levels showing no significant increase in Smad 1/5/8 phosphorylation. F Quantification of BMP-2-stimulated Western blot normalized against GAPDH levels showing a significant increase in p38 phosphorylation in both unstimulated and stimulated primary AF cells. n = 9 (each data point contains pooled IVD tissue samples from three mice containing six IVDs from each mouse; data points represent biological replicates of Flnb −/− protein lysates each normalized to a Flnb +/+ control. The dotted blue line in each graph represents the Flnb +/+ protein level, which is set to 1 upon normalization. Significance is determined by comparing Flnb −/− to Flnb +/+ ), NS not significant, * P < 0.05, ** P < 0.01, *** P < 0.001

Journal: Bone Research

Article Title: Intervertebral disc degeneration is rescued by TGFβ/BMP signaling modulation in an ex vivo filamin B mouse model

doi: 10.1038/s41413-022-00200-5

Figure Lengend Snippet: Isolated primary Flnb −/− AF cells exhibit increased TGFβ, ERK, and p38 signaling activation. a Western blots of protein lysates isolated from unstimulated primary AF cells (left column) and stimulated with 5 ng·mL −1 TGFβ-1 ligand for 30 min (right column) probed with antibodies against p-Smad 3, p-ERK1/2, and GAPDH. b Quantification of TGFβ-1-stimulated Western blot normalized against GAPDH levels showing a significant increase in Smad 3 phosphorylation in both unstimulated and stimulated primary AF cells. c Quantification of TGFβ-1-stimulated Western blot normalized against GAPDH levels showing no significant increase in ERK1/2 phosphorylation in either unstimulated or stimulated primary AF cells. d Western blots of isolated unstimulated primary AF cells (left column) and stimulated with 5 ng·mL −1 BMP-2 ligand for 30 min (right column) probed with antibodies against p-Smad 1/5/8, p-p38, and GAPDH. e Quantification of BMP-2-stimulated Western blot normalized against GAPDH levels showing no significant increase in Smad 1/5/8 phosphorylation. F Quantification of BMP-2-stimulated Western blot normalized against GAPDH levels showing a significant increase in p38 phosphorylation in both unstimulated and stimulated primary AF cells. n = 9 (each data point contains pooled IVD tissue samples from three mice containing six IVDs from each mouse; data points represent biological replicates of Flnb −/− protein lysates each normalized to a Flnb +/+ control. The dotted blue line in each graph represents the Flnb +/+ protein level, which is set to 1 upon normalization. Significance is determined by comparing Flnb −/− to Flnb +/+ ), NS not significant, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: MEFs were transfected with FLNB-GFP plasmid , Smad 1-Flag (Addgene 12622), Smad 6-HA (Addgene 14962), and Smad 7-HA (Addgene 11733) using the Neon Electroporation Transfection System (Thermo-Fisher).

Techniques: Isolation, Activation Assay, Western Blot, Phospho-proteomics, Control

$Higher levels of linker phosphorylated Smads 1 and 3 were found in the nucleus of Flnb − /− primary chondrocytes. a Western blot of Smad 1 linker phosphorylation levels in protein lysates isolated from Flnb +/+ and Flnb −/− IVDs. b Quantification of Western blot normalized to Actin levels showing a significant increase in Smad 1 linker phosphorylation. n = 3 (each data point contains six IVDs dissected from the thoracic area from a single mouse), NS not significant, * P value < 0.05. c – f Above: Western blots of Flnb +/+ and Flnb −/− cytoplasmic and nuclear protein lysate fractions isolated from primary sternal chondrocytes unstimulated and stimulated with 5 ng·mL −1 TGFβ-1 ligand and 10 ng·mL −1 BMP-2 ligand for 30 min probed with antibodies against Smad 3 linker phosphorylation at the S208 residue, Smad 1 linker phosphorylation at the S206 residue, GAPDH, and histone H3. Below: Quantification of TGFβ-1- and BMP-2-stimulated Western blots normalized against GAPDH (cytoplasmic fractions) and histone H3 (nuclear fractions) levels. The results showed a significant decrease in linker phosphorylated Smad 3 and Smad 1 levels in cytoplasmic fractions but a significant increase in nuclear fractions of Flnb −/− primary chondrocytes. n = 6 (All data points represent biological replicates of Flnb −/− protein lysates, each normalized to a Flnb +/+ control. The dotted blue line in each graph represents the Flnb +/+ protein level, which is set to 1 upon normalization. Significance is determined by comparing Flnb −/− to Flnb +/+ ), NS not significant, * P < 0.05, ** P < 0.01, *** P < 0.001$

Journal: Bone Research

Article Title: Intervertebral disc degeneration is rescued by TGFβ/BMP signaling modulation in an ex vivo filamin B mouse model

doi: 10.1038/s41413-022-00200-5

Figure Lengend Snippet: Higher levels of linker phosphorylated Smads 1 and 3 were found in the nucleus of Flnb − /− primary chondrocytes. a Western blot of Smad 1 linker phosphorylation levels in protein lysates isolated from Flnb +/+ and Flnb −/− IVDs. b Quantification of Western blot normalized to Actin levels showing a significant increase in Smad 1 linker phosphorylation. n = 3 (each data point contains six IVDs dissected from the thoracic area from a single mouse), NS not significant, * P value < 0.05. c – f Above: Western blots of Flnb +/+ and Flnb −/− cytoplasmic and nuclear protein lysate fractions isolated from primary sternal chondrocytes unstimulated and stimulated with 5 ng·mL −1 TGFβ-1 ligand and 10 ng·mL −1 BMP-2 ligand for 30 min probed with antibodies against Smad 3 linker phosphorylation at the S208 residue, Smad 1 linker phosphorylation at the S206 residue, GAPDH, and histone H3. Below: Quantification of TGFβ-1- and BMP-2-stimulated Western blots normalized against GAPDH (cytoplasmic fractions) and histone H3 (nuclear fractions) levels. The results showed a significant decrease in linker phosphorylated Smad 3 and Smad 1 levels in cytoplasmic fractions but a significant increase in nuclear fractions of Flnb −/− primary chondrocytes. n = 6 (All data points represent biological replicates of Flnb −/− protein lysates, each normalized to a Flnb +/+ control. The dotted blue line in each graph represents the Flnb +/+ protein level, which is set to 1 upon normalization. Significance is determined by comparing Flnb −/− to Flnb +/+ ), NS not significant, * P < 0.05, ** P < 0.01, *** P < 0.001

Techniques: Western Blot, Phospho-proteomics, Isolation, Residue, Control

Smad 1 degradation is less efficient in Flnb −/− -transfected MEFs due to FLNB interaction with i-Smads. a Western blots of Flnb +/+ and Flnb –/– MEF lysates nontransfected or transfected with Smad 1-Flag plasmid, ubiquitin-HA plasmid, or both. Blots were probed using antibodies against Flag, HA, and Actin. b Quantification of total Smad 1-Flag levels in Flnb +/+ versus Flnb –/– MEFs normalized to actin levels. The results show that more Smad 1-Flag plasmid remained after transfection and that the 24-h recovery period indicated inefficient degradation in the absence of FLNB. c Quantification of total Smad 1-Flag levels in Flnb +/+ versus Flnb –/– MEFs transfected with ubiquitin normalized to both Actin levels and Smad 1-Flag levels without ubiquitin transfection. The results again show that more Smad 1-Flag plasmid remained after ubiquitin transfection, indicating a lower efficiency in ubiquitinated degradation in the absence of FLNB. n = 3, * P < 0.05, *** P < 0.001. d Western blots of lysates isolated from wild-type MEFs transfected with FLNB-GFP and Smad 1-Flag plasmids immunoprecipitated using an antibody against GFP. FLNB western blot was probed using a GFP antibody, and Smad 1 western blot was probed using a Flag antibody. The results showed the detection of Smad 1-Flag protein when lysates were immunoprecipitated using a GFP antibody, confirming the in vitro interaction of FLNB and Smad 1. e Western blots of lysates isolated from wild-type MEFs transfected with FLNB-GFP and Smad 6-HA plasmids immunoprecipitated using an antibody against GFP. The results showed the detection of Smad 6-HA protein when lysates were immunoprecipitated using a GFP antibody, confirming the in vitro interaction of FLNB and Smad 6. f Western blots of lysates isolated from wild-type MEFs transfected with FLNB-GFP and Smad 7-HA plasmids immunoprecipitated using an antibody against GFP. The results showed the detection of Smad 7-HA protein when lysates were immunoprecipitated using a GFP antibody, confirming the in vitro interaction of FLNB and Smad 7. n = 3, WCL Whole Cell Lysate, IP Immunoprecipitate

Journal: Bone Research

Article Title: Intervertebral disc degeneration is rescued by TGFβ/BMP signaling modulation in an ex vivo filamin B mouse model

doi: 10.1038/s41413-022-00200-5

Figure Lengend Snippet: Smad 1 degradation is less efficient in Flnb −/− -transfected MEFs due to FLNB interaction with i-Smads. a Western blots of Flnb +/+ and Flnb –/– MEF lysates nontransfected or transfected with Smad 1-Flag plasmid, ubiquitin-HA plasmid, or both. Blots were probed using antibodies against Flag, HA, and Actin. b Quantification of total Smad 1-Flag levels in Flnb +/+ versus Flnb –/– MEFs normalized to actin levels. The results show that more Smad 1-Flag plasmid remained after transfection and that the 24-h recovery period indicated inefficient degradation in the absence of FLNB. c Quantification of total Smad 1-Flag levels in Flnb +/+ versus Flnb –/– MEFs transfected with ubiquitin normalized to both Actin levels and Smad 1-Flag levels without ubiquitin transfection. The results again show that more Smad 1-Flag plasmid remained after ubiquitin transfection, indicating a lower efficiency in ubiquitinated degradation in the absence of FLNB. n = 3, * P < 0.05, *** P < 0.001. d Western blots of lysates isolated from wild-type MEFs transfected with FLNB-GFP and Smad 1-Flag plasmids immunoprecipitated using an antibody against GFP. FLNB western blot was probed using a GFP antibody, and Smad 1 western blot was probed using a Flag antibody. The results showed the detection of Smad 1-Flag protein when lysates were immunoprecipitated using a GFP antibody, confirming the in vitro interaction of FLNB and Smad 1. e Western blots of lysates isolated from wild-type MEFs transfected with FLNB-GFP and Smad 6-HA plasmids immunoprecipitated using an antibody against GFP. The results showed the detection of Smad 6-HA protein when lysates were immunoprecipitated using a GFP antibody, confirming the in vitro interaction of FLNB and Smad 6. f Western blots of lysates isolated from wild-type MEFs transfected with FLNB-GFP and Smad 7-HA plasmids immunoprecipitated using an antibody against GFP. The results showed the detection of Smad 7-HA protein when lysates were immunoprecipitated using a GFP antibody, confirming the in vitro interaction of FLNB and Smad 7. n = 3, WCL Whole Cell Lysate, IP Immunoprecipitate

Techniques: Transfection, Western Blot, Plasmid Preparation, Ubiquitin Proteomics, Isolation, Immunoprecipitation, In Vitro

Protein and small molecule inhibitors used in cultured spine treatments

Journal: Bone Research

Article Title: Intervertebral disc degeneration is rescued by TGFβ/BMP signaling modulation in an ex vivo filamin B mouse model

doi: 10.1038/s41413-022-00200-5

Figure Lengend Snippet: Protein and small molecule inhibitors used in cultured spine treatments

Techniques: Cell Culture, Inhibition, Activity Assay, Ligand Binding Assay, Binding Assay, Phospho-proteomics

FLNB stabilizes i-Smad and Smad1 binding, inducing Smad1 degradation. Our model illustrates that under wild-type conditions ( a ), the Smad1 linker region is phosphorylated by TGFβ-activated ERK, which tags the molecule for degradation through the recruitment of Smad6, whose interaction with Smad1 is stabilized by FLNB. In the absence of FLNB ( b ), linker phosphorylated Smad1 is degraded less efficiently and allowed to induce increased transcription of nuclear targets

Journal: Bone Research

Article Title: Intervertebral disc degeneration is rescued by TGFβ/BMP signaling modulation in an ex vivo filamin B mouse model

doi: 10.1038/s41413-022-00200-5

Figure Lengend Snippet: FLNB stabilizes i-Smad and Smad1 binding, inducing Smad1 degradation. Our model illustrates that under wild-type conditions ( a ), the Smad1 linker region is phosphorylated by TGFβ-activated ERK, which tags the molecule for degradation through the recruitment of Smad6, whose interaction with Smad1 is stabilized by FLNB. In the absence of FLNB ( b ), linker phosphorylated Smad1 is degraded less efficiently and allowed to induce increased transcription of nuclear targets

Techniques: Binding Assay

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jeff wrana (Addgene inc)

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