shRNAs targeting mouse DDR2 RNA were purchase from Sigma (TRCN0000023594, TRCN0000361395). shRNA targeting human DDR2 RNA were purchase from Sigma (TRCN00000121117, TRCN0000121262, and TRCN0000121172). DDR2 cDNA from pDONR223-DDR2 (#23897, Addgene) was subcloned to PLX302 (#25896, Addgene) using Gateway cloning for lentiviral expression. Point mutants (K608A, Y740F) were generated using QuikChange II XL Site-Directed Mutagenesis Kit (#200521, Agilent). pWZL (control vector), pWZL Blast Twist ER (#18799, Addgene), pWZL Blast Snail ER (#18798, Addgene). Lentivirus expressing specific constructs was generated by transfecting HEK-293T cells in 6 well plates with a 1: 1: 0.1 ratio of lentiviral vector: pMD2.G: psPAX2 with TransIT-LT1 transfection reagent (Mirus). For retrovirus, a 1: 0.1: 1 ratio of retroviral vector: pCMV-VSV-G: pUMVC was used. After filtering through cellulose acetate membrane (0.45 μm, #28145–481, VWR), 250 ul of media with lentivirus were added to a 60mm dish of indicated cells with polybrene (8ug/ml) and selected with puromycin.
Pwzl blast twist er
Manufactured by Addgene
Sourced in United States
The PWZL Blast Twist ER is a laboratory equipment designed for centrifugation. It is capable of generating high-speed rotational forces to separate and isolate various biological components within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Pwzl blast snail er, by Addgene (3 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions)
Plx302, by Addgene (1 mentions)
Cellulose acetate membrane, by Avantor (1 mentions)
Quikchange 2 xl site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Pwzlblast gfp, by Addgene (1 mentions)
Mda mb 468, by American Type Culture Collection (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Skbr3, by American Type Culture Collection (1 mentions)
Quickchange 2 site directed mutagenesis kit, by Agilent Technologies (1 mentions)
4 protocols using pwzl blast twist er
1
Lentiviral and Retroviral Transduction Protocols
2
Inducible Snail and Twist Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
K562 cells were infected with retrovirus carrying either Snail-ER (Snail fused to ligand binding domain of estrogen receptor) (pWZL Blast Snail ER, Addgene plasmid #18798) or Twist1-ER constructs (pWZL Blast Twist ER, Addgene plasmid #18799). Both plasmids were a gift from Bob Weinberg 17 (link). The ER included in both constructs carried the G525R mutation, which abrogates its binding to 17β-estradiol while retaining its full sensitivity to the synthetic ligand 4'-hydroxytamoxifen. Infected cells were selected for resistance to blasticidin and were validated for the presence of Snail or Twist by RT-PCR and immunoblotting using anti-ERα antibodies.
3
Breast Cancer Cell Line Transfection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human breast cancer cell lines SKBR3, MDA-MB-468, MCF-7, and MDA-MB were purchased from ATCC. Cells were cultured in petri dishes with Dulbecco’s Modified Eagle Medium (DMEM; HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (HyClone, South Logan, UT, USA), and 1% penicillin/streptomycin (Gibco, Co Dublin, Ireland) in an atmosphere of 5% CO2 at 37 °C. Cells were passaged every 2–3 days using 0.25% Trypsin (Gibco, Co Dublin, Ireland). For plasmid transfection, pWZL Blast Twist ER, pWZL Blast Snail ER, and empty vector pWZL Blast GFP were obtained from Addgene (Cambridge, MA, USA). These plasmids were transfected into cells using Lipofectamine 3000 Reagent (Thermo Fisher, Waltham, MA, USA). The validation of transfection was conducted using quantitative RT-PCR analysis.
4
EpCAM expression regulation analysis
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!