pEGFP-C (Clontech) coding for Mus musculus (Mm)_Gli2 was kindly provided by Dr. Amin Liu (Department of Biology, Eberly College of Science, The Pennsylvania State University). Plasmids coding for Cerulean or myc-tagged RanWT or RanG19V were a gift from Dr. Kristen J. Verhey (Department of Cell and Developmental Biology, University of Michigan Medical School). Plasmids coding for myc-MBP or myc-MBP-M9M were kindly supplied by Dr. Yuh Min Chook (Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas). To generate the Gli2 NLS mutants, mutNLS-1 (R225A, K226A), mutNLS-2 (K557A, K578A) and the double mutant mutNLS-1+2 (R225A, K226A, K557A, K578A), we designed primers introducing the specified changes and performed site-directed mutagenesis using the Quick Change Site-Directed Mutagenesis Kit following the manufacturer’s instructions (Stratagene). Primers are available upon request.
Pegfp c
Manufactured by Takara Bio
Sourced in France
PEGFP-C is a plasmid vector that expresses the enhanced green fluorescent protein (EGFP) under the control of the cytomegalovirus (CMV) promoter. The EGFP coding sequence is fused to a polyethylene glycol (PEG) tag, which can be used to study the localization and trafficking of proteins in living cells.
Automatically generated - may contain errors
Lab products found in correlation
Pcdna3, by Thermo Fisher Scientific (2 mentions)
T4 dna ligase, by Thermo Fisher Scientific (2 mentions)
Pcdna 6.2 gw mir vector, by Thermo Fisher Scientific (1 mentions)
Mouse mtc panel 1, by Takara Bio (1 mentions)
Pcmv myc, by Takara Bio (1 mentions)
Pcmv ha, by Takara Bio (1 mentions)
Pgbkt7, by Takara Bio (1 mentions)
Molecular mass marker, by Thermo Fisher Scientific (1 mentions)
Liquid chromatography media, by GE Healthcare (1 mentions)
Pgex 4t 1, by GE Healthcare (1 mentions)
Restriction endonuclease, by Thermo Fisher Scientific (1 mentions)
Pmal c5x, by New England Biolabs (1 mentions)
Kits for dna isolation, by Thermo Fisher Scientific (1 mentions)
Phusion dna polymerase, by Thermo Fisher Scientific (1 mentions)
Anti flag antibody, by Cell Signaling Technology (1 mentions)
Pbind, by Promega (1 mentions)
Pcmv3tag, by Agilent Technologies (1 mentions)
Pcmvtnt, by Promega (1 mentions)
5 protocols using pegfp c
1
Generating Gli2 NLS Mutants in Mouse
2
Molecular Cloning of Expression Vectors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Various expression vectors were subcloned either to pcDNA3 (Invitrogen), pEGFPC (Clontech), or pcDNA6/BioEase (Invitrogen) using conventional molecular biology techniques and PCR14 (link),31 (link),32 (link). The sequence of Rab27a shRNA (5'-GCTGCCAATGGGACAAACATA-3') was used23 (link) and inserted into pcDNA 6.2-GW/miR vector (Invitrogen).
In the Ras experiments, we used H-Ras expression vector set (Clontech, pCMV-Ras vector, and pCMV-RasV12 vectors). GFP-ubiquitin was a gift from Nico Dantuma (Addgene plasmid # 11928)33 (link).
In the Ras experiments, we used H-Ras expression vector set (Clontech, pCMV-Ras vector, and pCMV-RasV12 vectors). GFP-ubiquitin was a gift from Nico Dantuma (Addgene plasmid # 11928)33 (link).
3
Cloning and Expressing Inserts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Inserts were amplified from kidney cDNA (Mouse MTC™ Panel I, Clontech, Palo Alto, CA). For yeast two-hybrid screening, inserts were cloned into the vector pGBKT7 (Clontech) in frame with the Gal4 DNA-binding domain. For expression in mammalian cells, cDNAs were cloned into pcDNA3.1 (Invitrogen, Carlsbad, CA), or vectors with various N-terminal tags (pCMV-Myc, pCMV-HA, or pEGFP-C; all from Clontech).
4
Bacterial Expression of Recombinant Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Restriction endonucleases, T4 DNA ligase, Phusion DNA polymerase, molecular mass markers, and kits for DNA isolation were obtained from Thermo Fisher Scientific (Moscow, Russia). The lysogeny broth Miller recipe (LB) medium was obtained from Amresco (Solon, OH, USA); liquid chromatography media from GE Healthcare (Moscow, Russia); reagents for agarose and polyacrylamide gel electrophoresis from Bio-Rad (Moscow, Russia); and general laboratory reagents from Merck (Moscow, Russia).
Gene cloning and recombinant protein production were carried out in Escherichia coli DH10B and Rosetta (DE3), respectively (Merck). Vectors for cloning and recombinant protein expression in E. coli included pMal-c5x (cloning with the NH2-terminal maltose-binding protein (MBP), New England Biolabs, Ipswich, MA, USA) and pGEX4T-1 (cloning with the NH2-terminal glutathione-S-transferase (GST), GE Healthcare). Anti-MBP antibody (#E8032S) was purchased from New England Biolabs, and anti-FLAG antibody (#14793) from Cell Signaling (Leiden, The Netherlands). The source of the green fluorescent protein coding sequence (eGFP) was the plasmid pEGFP-C, obtained from Clontech (Takara Bio Europe SAS, Saint-Germain-en-Laye, France).
Gene cloning and recombinant protein production were carried out in Escherichia coli DH10B and Rosetta (DE3), respectively (Merck). Vectors for cloning and recombinant protein expression in E. coli included pMal-c5x (cloning with the NH2-terminal maltose-binding protein (MBP), New England Biolabs, Ipswich, MA, USA) and pGEX4T-1 (cloning with the NH2-terminal glutathione-S-transferase (GST), GE Healthcare). Anti-MBP antibody (#E8032S) was purchased from New England Biolabs, and anti-FLAG antibody (#14793) from Cell Signaling (Leiden, The Netherlands). The source of the green fluorescent protein coding sequence (eGFP) was the plasmid pEGFP-C, obtained from Clontech (Takara Bio Europe SAS, Saint-Germain-en-Laye, France).
5
Construction of Recombinant Expression Vectors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Expression vectors were constructed as follows: myc-tagged human TSHZ1, TSHZ2, TSHZ3 and mutated or deleted TSHZ2 from pCMV-3Tag (Agilent Technologies); DsRed-tagged human TSHZ2 from pDsRed-monomer (Clontech); GAL4-fused TSHZ2 from pBIND (Promega); HA-tagged or FLAG-EGFP-tagged human GLI1 and GLI2 and FLAG-tagged human CtBP2 from pCMVTNT (Promega); and EGFP-tagged GLI1 and GLI2 from pEGFP-C (Clontech). Mutated and deleted TSHZ2 constructs were generated via PCR-amplification. Luciferase reporter vectors were constructed as follows: pG5TKluc from UAS-containing pG5luc (Promega) modified by the introduction of the thymidine kinase minimal promoter (TKmini); wtGBSx4TKLuc and mutGBSx4TKLuc from the luciferase reporter pGL3 (Promega) modified by the introduction of four copies of the wild-type or mutated GLI-binding site of the human CXCR4 promoter. Lentiviral vectors expressing either human TSHZ2, GLI1 or LacZ were constructed from the CSII-CMV-MCS-IRES2-Bsd plasmid, which was kindly provided by Dr. Hiroyuki Miyoshi (RIKEN BioResource Center, Japan).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!