cDNA for all 23 CRs was obtained from Addgene, except for cDNAs encoding CCR1, CCR9, XCR1, ACKR1, ACKR2, and ACKR5, which were bought from the Arizona State University. cDNAs encoding metabotropic glutamate receptor 1 (mGlu1R), α1b-AR, and AVPR1A were also purchased from Addgene. BRET sensor, GPCR-Rluc or GPCR-YFP, was produced by inserting DNAs encoding EYFP or Renilla luciferase (Rluc) in frame at the C-terminus of the above GPCRs into the Age I/Xba I sites, using IDTG (single-letter amino acid code) as spacer sequence between the GPCR C-termini and the BRET sensors. TRUPATH biosensors, Gαi1-Rluc8, Gβ3, and Gγ9-GFP2, were from Addgene deposited by the laboratory of Dr. Bryan Roth (32 (link)). All plasmid sequences were verified using Sanger sequencing.
Cdnas
Manufactured by Addgene
Sourced in United States
cDNAs, or complementary DNA molecules, are synthetic DNA sequences that are produced from mature mRNA templates. They represent the protein-coding regions of genes and are widely used in molecular biology research and applications.
Automatically generated - may contain errors
Lab products found in correlation
Mel624, by American Type Culture Collection (1 mentions)
Pcdna3.1 vector, by Thermo Fisher Scientific (1 mentions)
Ultracentrifugation, by Beckman Coulter (1 mentions)
Gp2 293 packaging cells, by Takara Bio (1 mentions)
C myc, by Addgene (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Polybrene, by Merck Group (1 mentions)
Anisomycin, by Merck Group (1 mentions)
Calcein red orange, by Thermo Fisher Scientific (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Alexis Biochemicals (1 mentions)
Thapsigargin tg, by Adipogen (1 mentions)
5 protocols using cdnas
1
GPCR Biosensor Plasmid Construction
2
Recombinant Protein Expression Strategies
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cDNAs encoding proteins in this study were purchased from Addgene or synthesized by Integrated DNA Technologies. All mutations were generated by PCR and tagged with either FLAG or HA or His, and cloned into vectors by Gibson assembly. pCDNA3.1(+) vector (Invitrogen) was used for transient expression; viral vectors (Clontech) for stable expression; and pET-28a (Novagen) for bacterial expression.
Wild-type, BRAF−/− and CRAF−/− MEFs were generated in previous study [58 (link), 59 (link)]. Melanoma cell lines: MeWo, A101D, Mel-624 were obtained from ATCC.
Wild-type, BRAF−/− and CRAF−/− MEFs were generated in previous study [58 (link), 59 (link)]. Melanoma cell lines: MeWo, A101D, Mel-624 were obtained from ATCC.
3
Generation of Stable Cell Lines Expressing p53
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For stable silencing of the endogenous mutant-p53 of MDA-MB-231 cells or the endogenous normal-p53 of HMLE cells, pLKO.1-Puro vector-based recombinant lentiviruses were generated, according to the method described previously.53 (link) For generation of shp53 cells stably expressing normal- or mutant-p53 proteins, pBabe-based retroviruses54 (link) were generated. cDNAs encoding V5-tagged normal-p53 protein and mutant-p53 proteins (R175H, R249S, R273H and R280K) were purchased from Addgene (cat# 22945, cat# 22936, cat# 22935, cat# 22934, and cat# 22933, respectively). Each of these DNA fragments was ligated into the SnaB1 site of pBabe-Hygro. For generation of HMLE cells stably expressing Snail, the retroviral plasmid vector (pBabe puro Snail) was purchased from Addgene (#23347). Production of recombinant retroviruses using Plat-E packaging cells, and their infection and selection of the infected cells, were performed as described previously.55 (link) During these experiments, we observed that expression of normal-p53 in shp53 cells using pLKO.1-based lentivirus or pcDNA-based plasmid caused cell senescence and death.
4
Reprogramming MEFs to iPSCs
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GP2-293 packaging cells (Clontech, Mountain View, CA, USA) were transfected with the pMX vectors harboring the human complementary DNAs (cDNAs) for Oct4 (POU5F1), Sox2, Klf4, and c-Myc (Addgene, Cambridge, MA, USA) and the VSV-G envelope vector using Lipofectamine 2000 transfection reagent (Invitrogen). Virus-containing supernatants were concentrated by ultracentrifugation (Beckman Coulter, Brea, CA, USA) at 25,000 rpm (rotor: SW32Ti) for 90 min. To generate iPSCs, MEFs or HFFs were seeded at 1 × 105 cells per well in six-well plates and then were transduced with virus at a multiplicity of infection of 1 in the presence of 8 μg/ml of polybrene (Sigma) on day 1. The MEFs or HFFs were trypsinized at day 4 or 5 after seeding, respectively, and were reseeded at a density of 3 × 104 cells per well in Matrigel-coated 12-well plates. On the next day, the medium was replaced with mESC or hESC medium with or without test chemicals, and the medium was changed every other day thereafter.
5
Fluorescent Biosensors in Hematopoietic Cells
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TALL-104 cells were obtained from the ATCC and cultured and transfected using nucleofection as described previously. 18 K562 cells were obtained from the ATCC and cultured in Iscove's modified Eagle's medium (MEM) supplemented with 10% heat-inactivated serum, glutamine, and penicillinstreptomycin, and were transfected using nucleofection according to the manufacturer's recommended protocol. CDNAs encoding EKAR, 19 CKAR, 2 DAGR, 2 DKAR, 20 and D3CPV 21 were obtained from Addgene (see Suppl. Table for a description of each sensor). The JNKAR construct 22 was provided by Dr. Jin Zhang. To generate polyclonal long-term transfectants, K562 cells were transiently transfected, then sorted on YPF fluorescence ~2 weeks and ~4 weeks after transfection. Experiments with transiently transfected TALL-104 cells were conducted in normal Ringer's solution. 18 All other experiments were conducted in complete medium. Phorbol 12-myristate 13-acetate (PMA) was from Alexis Biochemicals (San Diego, CA). Thapsigargin (TG) was from AdipoGen (San Diego, CA). Anisomycin was from Sigma Chemical (St. Louis, MO). Calcein red-orange and Far Red DDAO-SE were from Molecular Probes (Eugene, OR).
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