plasmids pcmv5 flag taz  (Addgene inc)

Journal: Cell reports

Article Title: A TAZ-AXL-ABL2 feed-forward signaling axis promotes lung adenocarcinoma brain metastasis

doi: 10.1016/j.celrep.2019.11.018

Figure Lengend Snippet: A) Immunoblots of PC9-BrM3 cells treated with 500 ng/mL recombinant human GAS6 for 1 h prior to immunoprecipitation (IP) with anti-ABL2 antibody (n=3). B) Immunoblots of AXL and p-AXL Y702 in PC9 cells treated with GNF-5 for 24 h. C) Immunoblots of PC9 cells transduced with shRNAs for scramble (SCR), ABL1, ABL2, and ABL1/ABL2 double knockdown (AA) (n=3). D) Schematic of AXL and ABL2 protein structural domains. E) Co-IP of indicated proteins in 293T cells co-transfected with FLAG-AXL WT or phospho-mutants and WT ABL2-GFP plasmids as indicated. Cells were serum-starved for 1 hr prior to addition of 500 ng/mL human GAS6 for 1 h. F) Immunoblots of 293T cells co-transfected with FLAG-AXL and ABL2-GFP wild-type (WT), R198K (SH2-dead) or K317M (kinase dead) mutants. Cells were serum-starved for 1 h prior to treatment with 500 ng/mL GAS6 for 1 h. G) Immunoblot of PC9 cells transduced with inducible active ABL2PP treated with dox for 24 h. H) Immunoblots of PC9-BrM3 cells treated ± 5 uM BGB324 for 24 h. I) Immunoblots of PC9-BrM3 cells transduced with shNTC or shAXL (n=3). WCL: whole cell lysate.

Article Snippet: The 3XFlag pCMV5-TOPO-TAZ wild type (WT) plasmid was a gift from Jeff Wrana (Addgene plasmid # 24809) ( Varelas et al., 2008 ). pWZL-Neo-Myr-Flag-AXL was a gift from William Hahn & Jean Zhao (Addgene plasmid # 20428) ( Boehm et al., 2007 ). pN1-ABL2-eGFP (also known as pN1-ARG-eGFP) was a gift from Anthony Koleske (Yale University, New Haven, CT, USA).

Techniques: Western Blot, Recombinant, Immunoprecipitation, Transduction, Knockdown, Co-Immunoprecipitation Assay, Transfection

Journal: Cell reports

Article Title: A TAZ-AXL-ABL2 feed-forward signaling axis promotes lung adenocarcinoma brain metastasis

doi: 10.1016/j.celrep.2019.11.018

Figure Lengend Snippet: A) Co-IP of PC9-Tet-ABL2PP cells transfected with FLAG-TAZ wild-type (WT) and treated with dox for 1 hr prior to anti-FLAG pulldown. B) PC9-BrM3 cells were transfected with FLAG-TAZ-WT or FLAG-TAZ-Y321F, and following anti-FLAG pulldown, co-IP proteins were immunoblotted with indicated antibodies. C) Co-IP of indicated proteins in 293T cells co-transfected with TAZ-FLAG and ABL2-GFP WT, R198K or K317M mutants. D) Nuclear fractionation of PC9-Tet-ABL2PP cells treated with dox for indicated time points. GAPDH and Lamin B1 used for cytosolic and nuclear markers, respectively (n=3). E) Co-IP of PC9-BrM3 cells with antibodies against ABL1, ABL2, or IgG control. WCL: whole cell lysate.

Article Snippet: The 3XFlag pCMV5-TOPO-TAZ wild type (WT) plasmid was a gift from Jeff Wrana (Addgene plasmid # 24809) ( Varelas et al., 2008 ). pWZL-Neo-Myr-Flag-AXL was a gift from William Hahn & Jean Zhao (Addgene plasmid # 20428) ( Boehm et al., 2007 ). pN1-ABL2-eGFP (also known as pN1-ARG-eGFP) was a gift from Anthony Koleske (Yale University, New Haven, CT, USA).

Techniques: Co-Immunoprecipitation Assay, Transfection, Fractionation, Control

Journal: Cell reports

Article Title: A TAZ-AXL-ABL2 feed-forward signaling axis promotes lung adenocarcinoma brain metastasis

doi: 10.1016/j.celrep.2019.11.018

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The 3XFlag pCMV5-TOPO-TAZ wild type (WT) plasmid was a gift from Jeff Wrana (Addgene plasmid # 24809) ( Varelas et al., 2008 ). pWZL-Neo-Myr-Flag-AXL was a gift from William Hahn & Jean Zhao (Addgene plasmid # 20428) ( Boehm et al., 2007 ). pN1-ABL2-eGFP (also known as pN1-ARG-eGFP) was a gift from Anthony Koleske (Yale University, New Haven, CT, USA).

Techniques: Western Blot, Immunofluorescence, Recombinant, In Vivo, In Vitro, Mutagenesis, Caspase-Glo Assay, RNA Sequencing, shRNA, Control, Plasmid Preparation, Software

Journal: Cell reports

Article Title: A TAZ-AXL-ABL2 feed-forward signaling axis promotes lung adenocarcinoma brain metastasis

doi: 10.1016/j.celrep.2019.11.018

Figure Lengend Snippet: A) Representative images (day 30 post-injection) and B) analysis of brain metastasis-free survival (BMFS) in mice injected intracardially with PC9-pFuLT or pFuLT-Tet-TAZ4SA-expressing cells. Mice were given dox water for the duration of the study. Statistical analysis calculated by Log-rank (Mantel-Cox) test. Parental (n=13), TAZ4SA (n=17). C-D) Quantitative analysis (day 30 post-injection) of brain-metastatic index in mice injected intracardially with C) PC9-pFuLT (n=13) vs. PC9-pFuLT-Tet-TAZ4SA (n=17) or D) HCC4006-pFuLT (n=9) vs HCC4006-pFuLT-Tet-TAZ4SA (n=10) cells. Statistical analysis calculated by unpaired two-tailed t test. ** p-value < 0.01. E) Representative images of ex vivo mouse brains on day 32 post-injection with HCC4006-pFuLT or HCC4006-Tet-TAZ4SA cells. F) Diagram of iterative derivation of brain-metastatic cell lines. G) GSEA plot of TAZ4SA signature in PC9 parental vs PC9-BrM3 RNA-seq dataset. NES=normalized enrichment score. H) Venn diagram of overlapping transcripts upregulated in PC9-BrM3 and PC9-TAZ4SA cells. I) Representative images (day 42 post-injection) and J) BMFS in mice injected intracardially with PC9-BrM3 cells expressing non-target control (shNTC, n=10) or shTAZ (clone #73, n=10). K) Immunoblot to evaluate TAZ shRNA knockdown in PC9-BrM3 cells. Actin was used for protein loading control.

Article Snippet: Recombinant DNA & Plasmids pLKO-puro Non-Target shRNA Control Sigma Mission TRC1 SHC016-1EA pLKO-puro shAXL 1040 Sigma Mission TRC1 TRCN0000001040 pLKO-puro shTAZ 70 Sigma Mission TRC1 TRCN0000019470 pLKO-puro shTAZ 71 Sigma Mission TRC1 TRCN0000019471 pLKO-puro shTAZ 73 Sigma Mission TRC1 TRCN0000019473 TetO-FUW-pgk-puro ( Chowdhury et al., 2016 ) Addgene #85747; RRID:Addgene_85747 pLVX-Tight-Puro Tet-On Vector Xaralabos Varelas, Boston University, Boston, MA, USA N/A pLVX-TP-3F-TAZ4SA Xaralabos Varelas, Boston University, Boston, MA, USA N/A Scramble control shRNA ( Gu et al, 2016 ) N/A ABL1 shRNA ( Gu et al, 2016 ) N/A ABL2 shRNA ( Gu et al, 2016 ) N/A 3XFlag pCMV5-TOPO TAZ wild type ( Varelas et al., 2008 ) Addgene #24809; RRID:Addgene_24809 3XFlag pCMV5-TOPO TAZ Y321F This paper N/A Tet-pLKO-puro ( Wiederschain et al., 2009 ) Addgene #21915; RRID:Addgene_21915 Tet-pLKO-shNTC-puro This paper N/A Tet-pLKO-shAXL-puro This paper N/A, shAXL clone TRCN0000001040 Tet-pLKO-shTAZ-puro This paper N/A, shTAZ clone TRCN0000019470 N174-MCS http://n2t.net/addgene:81061 Addgene #81061; RRID:Addgene_81061 phL1A-pcDNA3 ( Hlavin and Lemmon, 1991 ) Addgene #12307; RRID:Addgene_12307 N174-L1CAM This paper N/A pN1-eGFP Anthony Koleske, Yale University, New Haven, CT, USA N/A pN1-ABL2-eGFP WT Anthony Koleske, Yale University, New Haven, CT, USA N/A pN1-ABL2-eGFP R198K This paper N/A pN1-ABL2-eGFP K317M This paper N/A pWZL Neo Myr Flag AXL ( Boehm et al., 2007 ) Addgene #20428; RRID:Addgene_20428 pWZL Neo Myr Flag AXL Y779F This paper N/A pWZL Neo Myr Flag AXL Y821F This paper N/A pWZL Neo Myr Flag AXL Y830F This paper N/A pWZL Neo Myr Flag AXL Y866F This paper N/A Software and Algorithms Prism 6 and 8 Graphpad http://graphpad.com/scientific-software/prism ImageJ ( Schneider et al., 2012 ) http://imagej.nih.gov RStudio R Foundation for Statistical Computing http://rstudio.com CBLigand Online BBB Predictor Tool v0.90 ( Liu et al., 2014 ) http://cbligand.org Living Image Perkin Elmer http://perkinelmer.com GSEA 3.0 ( Subramanian et al., 2005 ) http://software.broadinstitute.org/gsea KM Plotter ( Gyorffy et al., 2013 ) http://kmplot.com CBioPortal v3.1.1 ( Cerami et al., 2012 ; Gao et al., 2013 ) http://cbioportal.org TrimGalore v0.4.4 Babraham Bioinformatics http://github.com/FelixKrueger/TrimGalore STAR aligner ( Dobin et al., 2013 ) http://github.com/alexdobin/STAR HTSeq-count ( Anders et al., 2015 ) http://htseq.readthedocs.io DESeq2 v2.12 ( Love et al., 2014 ) http://bioconductor.org/packages/release/bioc/html/DESeq2.html Hisat2 v2.1.0 ( Kim et al., 2019 ) http://ccb.jhu.edu/software/hisat2/index.shtml Open in a separate window KEY RESOURCES TABLE

Techniques: Injection, Expressing, Two Tailed Test, Ex Vivo, RNA Sequencing, Control, Western Blot, shRNA, Knockdown

Journal: Cell reports

Article Title: A TAZ-AXL-ABL2 feed-forward signaling axis promotes lung adenocarcinoma brain metastasis

doi: 10.1016/j.celrep.2019.11.018

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Recombinant DNA & Plasmids pLKO-puro Non-Target shRNA Control Sigma Mission TRC1 SHC016-1EA pLKO-puro shAXL 1040 Sigma Mission TRC1 TRCN0000001040 pLKO-puro shTAZ 70 Sigma Mission TRC1 TRCN0000019470 pLKO-puro shTAZ 71 Sigma Mission TRC1 TRCN0000019471 pLKO-puro shTAZ 73 Sigma Mission TRC1 TRCN0000019473 TetO-FUW-pgk-puro ( Chowdhury et al., 2016 ) Addgene #85747; RRID:Addgene_85747 pLVX-Tight-Puro Tet-On Vector Xaralabos Varelas, Boston University, Boston, MA, USA N/A pLVX-TP-3F-TAZ4SA Xaralabos Varelas, Boston University, Boston, MA, USA N/A Scramble control shRNA ( Gu et al, 2016 ) N/A ABL1 shRNA ( Gu et al, 2016 ) N/A ABL2 shRNA ( Gu et al, 2016 ) N/A 3XFlag pCMV5-TOPO TAZ wild type ( Varelas et al., 2008 ) Addgene #24809; RRID:Addgene_24809 3XFlag pCMV5-TOPO TAZ Y321F This paper N/A Tet-pLKO-puro ( Wiederschain et al., 2009 ) Addgene #21915; RRID:Addgene_21915 Tet-pLKO-shNTC-puro This paper N/A Tet-pLKO-shAXL-puro This paper N/A, shAXL clone TRCN0000001040 Tet-pLKO-shTAZ-puro This paper N/A, shTAZ clone TRCN0000019470 N174-MCS http://n2t.net/addgene:81061 Addgene #81061; RRID:Addgene_81061 phL1A-pcDNA3 ( Hlavin and Lemmon, 1991 ) Addgene #12307; RRID:Addgene_12307 N174-L1CAM This paper N/A pN1-eGFP Anthony Koleske, Yale University, New Haven, CT, USA N/A pN1-ABL2-eGFP WT Anthony Koleske, Yale University, New Haven, CT, USA N/A pN1-ABL2-eGFP R198K This paper N/A pN1-ABL2-eGFP K317M This paper N/A pWZL Neo Myr Flag AXL ( Boehm et al., 2007 ) Addgene #20428; RRID:Addgene_20428 pWZL Neo Myr Flag AXL Y779F This paper N/A pWZL Neo Myr Flag AXL Y821F This paper N/A pWZL Neo Myr Flag AXL Y830F This paper N/A pWZL Neo Myr Flag AXL Y866F This paper N/A Software and Algorithms Prism 6 and 8 Graphpad http://graphpad.com/scientific-software/prism ImageJ ( Schneider et al., 2012 ) http://imagej.nih.gov RStudio R Foundation for Statistical Computing http://rstudio.com CBLigand Online BBB Predictor Tool v0.90 ( Liu et al., 2014 ) http://cbligand.org Living Image Perkin Elmer http://perkinelmer.com GSEA 3.0 ( Subramanian et al., 2005 ) http://software.broadinstitute.org/gsea KM Plotter ( Gyorffy et al., 2013 ) http://kmplot.com CBioPortal v3.1.1 ( Cerami et al., 2012 ; Gao et al., 2013 ) http://cbioportal.org TrimGalore v0.4.4 Babraham Bioinformatics http://github.com/FelixKrueger/TrimGalore STAR aligner ( Dobin et al., 2013 ) http://github.com/alexdobin/STAR HTSeq-count ( Anders et al., 2015 ) http://htseq.readthedocs.io DESeq2 v2.12 ( Love et al., 2014 ) http://bioconductor.org/packages/release/bioc/html/DESeq2.html Hisat2 v2.1.0 ( Kim et al., 2019 ) http://ccb.jhu.edu/software/hisat2/index.shtml Open in a separate window KEY RESOURCES TABLE

Techniques: Western Blot, Immunofluorescence, Recombinant, In Vivo, In Vitro, Mutagenesis, Caspase-Glo Assay, RNA Sequencing, shRNA, Control, Plasmid Preparation, Software

Journal: Oncotarget

Article Title: POPX2 is a novel LATS phosphatase that regulates the Hippo pathway

doi: 10.18632/oncotarget.26689

Figure Lengend Snippet: Lysates from HEK293 cells expressing GST-vector or GST-POPX2 together with ( A ) Flag-NDR1, ( B ) Myc-MOB1, ( C ) Flag-MST1, ( D ) Flag-LATS1, ( E ) Flag-YAP, and ( F ) Flag-TAZ were subjected to immunoprecipitation with glutathione sepharose beads and western analysis. Proteins associated with GST-POPX2 were detected by Western blotting (WB) with either anti-Flag or anti-Myc antibodies.

Article Snippet: The following plasmids were from Addgene: pPS2977-NDR1 (from Pamela Silver, Addgene plasmid # 8927), pClneoMyc human MOB1 (from Yutaka Hata, Addgene plasmid # 37024), pcDNA3 Lats1 (Nigg HS189) (from Erich Nigg, Addgene plasmid # 41156, 3XFlag pCMV5-TOPO TAZ WT (from Jeff Wrana, Addgene plasmid # 24809).

Techniques: Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: Oncotarget

Article Title: POPX2 is a novel LATS phosphatase that regulates the Hippo pathway

doi: 10.18632/oncotarget.26689

Figure Lengend Snippet: ( A ) Endogenous MST1 from HEK293 cells was immunoprecipitated. The IP complexes were separated by SDS PAGE, followed by Western analysis using POPX2 antibody. The Western blot reveals endogenous MST1-POPX2 interaction. ( B ) Similar experiment as in (A) was performed using LATS1 antibody for immunoprecipitation. The Western blot reveals endogenous LATS1-POPX2 interaction. ( C ) Reciprocal endogenous POPX2 immunoprecipitation followed by Western analysis using MST1 and LATS1 antibodies reveal endogenous POPX2-MST1 and POPX2-LATS1 interactions. Endogenous immunoprecipitation experiments are representative of 2 independent experiments; representative images were shown.

Article Snippet: The following plasmids were from Addgene: pPS2977-NDR1 (from Pamela Silver, Addgene plasmid # 8927), pClneoMyc human MOB1 (from Yutaka Hata, Addgene plasmid # 37024), pcDNA3 Lats1 (Nigg HS189) (from Erich Nigg, Addgene plasmid # 41156, 3XFlag pCMV5-TOPO TAZ WT (from Jeff Wrana, Addgene plasmid # 24809).

Techniques: Immunoprecipitation, SDS Page, Western Blot

Journal: Oncotarget

Article Title: POPX2 is a novel LATS phosphatase that regulates the Hippo pathway

doi: 10.18632/oncotarget.26689

Figure Lengend Snippet: ( A ) In vitro phosphatase assay. Bacterially expressed POPX2 (4 µg and 8 µg) was incubated with phospho-Flag-MST1 for 45 mins at 30°C. The reaction mixtures were subjected to SDS-PAGE and Western analysis using phospho-MST1-Thr183 antibodies. Calf-intestinal phosphatase (CIP) was used as a negative control. ( B ) HEK293 cells were transfected with GFP-POPX2 or GFP-Flag vector constructs. The cells were treated with okadaic acid (1 µM) for 1 hr to inhibit PP2A and induce MST1 autophosphorylation. Cell lysates were separated on SDS-PAGE and subjected to Western analysis to detect phospho-MST1-Thr183. ( C ) In vitro phosphatase assay. Bacterially expressed POPX2 was incubated with phospho-Flag-LATS1 instead of Flag-MST1 as in (A). The reaction mixtures were subjected to SDS-PAGE and Western analysis using phospho-LATS1-Thr1079 antibodies. Calf-intestinal phosphatase (CIP) was used as a negative control. CIP can remove phosphate group from phospho-tyrosine, serine and threonine, but with a preference for phospho-tyrosine. In our experiment, CIP was used as a negative control because when CIP was used in similar experimental condition as POPX2 for phosphatase assays, it was found not as efficient as POXP2 in dephosphorylating LATS1. ( D ) Densitometry quantification of (C) from three independent experiments. POPX2 dephosphorylates LATS1 in a dose dependent manner. ( E ) HEK293 cells were transfected with Flag-LATS1 + Myc-MOB1 together with GST-vector, GST-PP1, GST-PP2A or GST-POPX2. Cell lysates were harvested and separated by SDS-PAGE and subjected to Western analysis. Equal amounts of total protein lysates were loaded into each well of the gel. Total LATS1 (left panel) and pLATS1 (right panel) were probed on separate membranes. ( F ) Densitometry quantification of phospho-LATS1. Three independent experiments were performed. Error bars represent standard deviation. Student t -test was performed to determine statistical significances.

Article Snippet: The following plasmids were from Addgene: pPS2977-NDR1 (from Pamela Silver, Addgene plasmid # 8927), pClneoMyc human MOB1 (from Yutaka Hata, Addgene plasmid # 37024), pcDNA3 Lats1 (Nigg HS189) (from Erich Nigg, Addgene plasmid # 41156, 3XFlag pCMV5-TOPO TAZ WT (from Jeff Wrana, Addgene plasmid # 24809).

Techniques: In Vitro, Phosphatase Assay, Incubation, SDS Page, Western Blot, Negative Control, Transfection, Plasmid Preparation, Construct, Standard Deviation

Journal: Oncotarget

Article Title: POPX2 is a novel LATS phosphatase that regulates the Hippo pathway

doi: 10.18632/oncotarget.26689

Figure Lengend Snippet: ( A ) High levels of POPX2 lead to dephosphorylation of LATS1 on Thr1079 and inactivation of LATS1 kinase activity. Absence of LATS1 activity prevents YAP/TAZ degradation and promotes YAP/TAZ nuclear translocation. Active YAP/TAZ will then promote transcription of target genes. ( B ) When POPX2 is absent, LATS1 is phosphorylated and activated. Active LATS1 is capable of phosphorylating YAP/TAZ, which are then targeted for degradation by proteosomes.

Article Snippet: The following plasmids were from Addgene: pPS2977-NDR1 (from Pamela Silver, Addgene plasmid # 8927), pClneoMyc human MOB1 (from Yutaka Hata, Addgene plasmid # 37024), pcDNA3 Lats1 (Nigg HS189) (from Erich Nigg, Addgene plasmid # 41156, 3XFlag pCMV5-TOPO TAZ WT (from Jeff Wrana, Addgene plasmid # 24809).

Techniques: De-Phosphorylation Assay, Activity Assay, Translocation Assay