Standard recombinant DNA techniques were used to construct pCS2/FLAG-rat Fz2 (FLAG-Fz2) and pPGK-neo/Wnt5a. pSuper-retro-GFP-Neo-shWnt5a, which was used for the generation of cells stably expressing Wnt5a shRNA, was generated by inserting small hairpin RNA against Wnt5a (5′-GTGGATAACACCTCTGTT-3′) into the pSuper-retro-GFP-Neo vector (Oligo Engine, Seattle, WA). The small interfering RNAs (siRNAs) used in this study are listed in Table S1 . Wnt5a was purified to near homogeneity using three chromatography steps as described previously16 (link)55 (link). Anti-Wnt3a, anti-Wnt5a/b, anti-Src, anti-Fyn, anti-Yes, anti-phospho-Src family (Tyr416), anti-AKT, anti-phospho-AKT (Ser473), anti-phospho-PKC (pan) (Ser660), anti-SAPK/JNK, and anti-phospho-SAPK/JNK (Thr183/Tyr185) antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-Rac1, anti-EEA1, anti-HSP90, anti-PKCα, and anti-Clathrin antibodies were from BD Biosciences (San Jose, CA). Anti-FLAG M2 and anti-Ror2 antibodies were from Sigma-Aldrich (Steinheim, Germany) and R&D Systems (Minneapolis, MN), respectively.
Psuper retro neo gfp vector
Manufactured by Oligoengine
Sourced in United States
The PSuper retro-neo-GFP vector is a plasmid-based system designed for the expression of short hairpin RNA (shRNA) constructs in mammalian cells. The vector contains the necessary elements for retroviral production and integration, as well as the neomycin resistance gene for selection of stably transfected cells. The green fluorescent protein (GFP) marker is included to facilitate the monitoring of vector expression.
Automatically generated - may contain errors
Lab products found in correlation
Puromycin, by Thermo Fisher Scientific (2 mentions)
Sb202190, by Merck Group (2 mentions)
Bms 345541, by Abcam (2 mentions)
Lipofectamine 2000 and 3000, by Thermo Fisher Scientific (2 mentions)
Lenticrispr v2, by Addgene (2 mentions)
Sp600125, by Merck Group (2 mentions)
Anti phospho akt ser473, by Cell Signaling Technology (1 mentions)
Anti rac1, by BD (1 mentions)
Anti pkc α, by BD (1 mentions)
Psuper retro neo gfp, by Oligoengine (1 mentions)
Anti phospho sapk jnk thr183 tyr185, by Cell Signaling Technology (1 mentions)
Anti clathrin, by BD (1 mentions)
Anti sapk jnk, by Cell Signaling Technology (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Anti hsp90, by BD (1 mentions)
Anti eea1, by BD (1 mentions)
5 protocols using psuper retro neo gfp vector
1
Purification and Manipulation of Wnt Signaling
2
shRNA Cloning and Target Sequences
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The used shRNAs were cloned into the pSUPER.retro.gfp+neo vector (Oligoengine, Seattle, WA, USA). The following already published target sequences were used: SEPT7 CTTGCAGCTGTGACTTATA [61 (link)] and control GATCTGATCGACACTGTAA.
3
Efficient PLSCR1 Protein Silencing
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Four different shRNA PLSCR1 constructs were obtained by sub-cloning the double-stranded 64-mer oligonucleotide containing the PLSCR1 target sequences (A: 5′-GGACCTCCAGGATATAGTG-3′; B: 5′-CTCTGGAGAGACCACTAAG-3′; C:5′-AGTCTCCTCAGGAAATCTG-3′) or the mismatched sequence (MIS: 5′-GGACGTCCTGGATTTAGTG-3′) into the pSUPER.retro.neo+GFP vector (pSUPER; OligoEngine). Infection was performed as described above. Immunoblotting analysis of transfected Phoenix cells identified the construct shPLSCR1A as the most efficient in protein silencing, therefore we selected this to perform all subsequent experiments. Upon infection, Mino cells were selected with G418 (1 mg/mL) and the infection efficiency was checked through the detection of GFP expression by flow cytometry (97% positive cells).
4
Knocking Down TAK1 in NMuMG Cells
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For knocking down TAK1 in NMuMG cells, pSuper retro-neo-GFP vector (OligoEngine, Seattle, WA) was used to construct shTAK1 plasmid that targets 3’-UTR of mouse TAK1 gene. Flag-tagged mouse TAK1FL or TAK1∆E12 plasmids were constructed by insert TAK1 cDNA from MGC clone 5989 (Image: 3499247) or HA-TAK1 (gift from Dr. K. Matsumoto) (10 (link)) to pLNCX vector, respectively. For CRISPR/Cas9 mediated editing of the TAK1 gene, plasmid lentiCRISPRv2 (Addgene 52961) was used to deliver individual sgRNAs (single guide RNA). Sequences of primers used in the construction of shRNA, cDNA and CRISPR/Cas9 vectors are listed in Supplementary Table S1 . Stocks of lentiviruses were made after co-transfecting lentiCRISPRv2, pVSVG and psPAX2 plasmids into HEK293T cells. Lipofectamine 2000 and 3000 (Life Technologies, Grand island, NY) was used to transfect plasmid DNA according to the manufacturer’s instructions. G418 or puromycin (Life Technologies) was used to select stable clones. JNK inhibitor, SP600125, and p38 inhibitor, SB202190 were obtained from EMD Millipore (Burlington, MA), NF-kB inhibitor, BMS345541, was obtained from Abcam (Cambridge, MA)
5
Knocking Down TAK1 in NMuMG Cells
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