Log In Sign up
The largest database of trusted experimental protocols

Pbabe puro hras v12

Manufactured by Addgene
Visit Supplier
Sourced in United States

The PBabe-puro HRAS V12 is a plasmid construct that contains the oncogenic HRAS V12 gene. This construct is commonly used for the expression of the constitutively active HRAS protein in cell culture experiments.

Automatically generated - may contain errors

Lab products found in correlation

Pbabe puro hras v12 s35, by Addgene (2 mentions) Pbabe puro, by Addgene (2 mentions) Px459 v2, by Addgene (1 mentions) Pbabe krasg12d, by Addgene (1 mentions) Hras v12, by Addgene (1 mentions) Pgl4.22 vector, by Promega (1 mentions) Pcdna3.1 flag claspin, by Addgene (1 mentions) Pcdna4 flag timeless, by Addgene (1 mentions) Pmaxgfp plasmid, by Lonza (1 mentions) Pbabe puro braf v600e, by Addgene (1 mentions) Mycoalert mycoplasma detection kit, by Lonza (1 mentions) Sk mel 28, by American Type Culture Collection (1 mentions) Sk mel 2, by American Type Culture Collection (1 mentions) Pbabe puro mek dd, by Addgene (1 mentions) Polybrene, by Merck Group (1 mentions) Pvsv g, by Takara Bio (1 mentions) Calphos mammalian transfection kit, by Takara Bio (1 mentions) Pbabe puro k ras v12, by Addgene (1 mentions) Puromycin, by InvivoGen (1 mentions) Pwzl hygro h ras v12, by Addgene (1 mentions)

7 protocols using pbabe puro hras v12

1

Plasmids for Cell Engineering

Check if the same lab product or an alternative is used in the 5 most similar protocols
The plasmids pBabe-puro (Addgene # 1764) were a gift from Hartmut Land and Jay Morgenstern, pBabe-puro HRAS V12 (Addgene # 39526), were a gift from Julian Downward and HRAS V12 (Addgene # 9051) was a gift from William Hahn, pbabe - KRAS G12D (Addgene # 58902) was a gift from Channing Der pBabe-puro HRAS V12 S35 (Addgene # 18746) was a gift from Jay Morgenstern, pBabe-puro HRAS V12 C40 (Addgene # 18747) and pBabe-puro HRAS V12 G37 (Addgene # 18745) was a gift from Scott Lowe S100A10 siRNA was purchased as a pre-designed sequence from Ambion (4392420). The pKRAS-gRNA1-px459-V2 plasmid was made by cloning the annealed oligos: 5′- CAC CGA ATA TAA ACT TGT GGT AGT-3′ and 5′-AAA CAC TAC CAC AAG TTT ATA TTC-3′ into px459-V2 from Addgene (62988) and the pKRAS-gRNA2-px459-V2 was made by cloning the annealed oligos: 5′-CAC CGA AAC TTG TGG TAG TTG GAG C-3′ and 5′-AAA CGC TCC AAC TAC CAC AAG TTT C-3′ into px459-V2 from Addgene. The pGL4.22- S100A10-promoter luciferase reporter plasmid was constructed by cutting the S100A10-promoter from the pCAT-S100A10-promoter plasmid and cloning it into the Kpn I/Sac I restriction sites of pGL4.22 vector (Promega).
Madureira P.A., Bharadwaj A.G., Bydoun M., Garant K., O'Connell P., Lee P, & Waisman D.M. (2016). Cell surface protease activation during RAS transformation: Critical role of the plasminogen receptor, S100A10. Oncotarget, 7(30), 47720-47737.
+ Open protocol
+ Expand
2

Transfection and Viral Concentration

Check if the same lab product or an alternative is used in the 5 most similar protocols
293GPG cells were transfected with the pBABE puro H-RasV12 (Addgene, Plasmid #9051) viral vector. Viral suspensions were collected at five time points (at 2-day intervals). After the final collection, the viral suspension was filtered through a 0.45-μm filter and concentrated using the Retro-X concentrator system (Thermo Fisher Scientific). The concentrated virus was suspended in TNE buffer (50 mM Tris, 130 mM NaCl, and 1 mM ethylenediaminetetraacetic acid).
Lee S.H., Yang J.H., Park U.H., Choi H., Kim Y.S., Yoon B.E., Han H.J., Kim H.T., Um S.J, & Kim E.J. (2023). SIRT1 ubiquitination is regulated by opposing activities of APC/C-Cdh1 and AROS during stress-induced premature senescence. Experimental & Molecular Medicine, 55(6), 1232-1246.
+ Open protocol
+ Expand
3

Ras and Cyclin E Overexpression Protocols

Check if the same lab product or an alternative is used in the 5 most similar protocols
pBABE-Puro H-RasV12 (n°12545 Addgene) or Cyclin E and corresponding empty vectors were used to infect either BJ hTERT cells or U2OS, respectively. Lentiviruses were produced by the vectorology facility (BioCampus Montpellier) according to standard protocols76 (link). Plasmid transfection in BJ-Rasv12 cells was performed using Viromer Yellow (Lipocalix) according to the manufacturer’s instructions. U2OS-CycE transfections were performed using Interferin (Polyplus-transfection, Illkirch, France) according to the manufacturer’s instructions. As a control, pMax-GFP plasmid (Lonza) was used and to overexpressed Claspin and Timeless pcDNA3.1-Flag-claspin (Addgene no. 12659) and pcDNA 4-Flag-Timeless (Addgene no. 22887) plasmids were used.
Bianco J.N., Bergoglio V., Lin Y.L., Pillaire M.J., Schmitz A.L., Gilhodes J., Lusque A., Mazières J., Lacroix-Triki M., Roumeliotis T.I., Choudhary J., Moreaux J., Hoffmann J.S., Tourrière H, & Pasero P. (2019). Overexpression of Claspin and Timeless protects cancer cells from replication stress in a checkpoint-independent manner. Nature Communications, 10, 910.
+ Open protocol
+ Expand
4

Melanoma Cell Lines Characterization

Check if the same lab product or an alternative is used in the 5 most similar protocols
SKMEL-2, SKMEL-28, MeWo, and A375 cells were purchased from American Type Culture Collection (ATCC) and grown as recommended. Neonatal primary human melanocytes were purchased from Life Technologies and grown as recommended by the supplier. All short-term melanoma cultures (YUGASP, YUHEF, YUTOGS, and YUVON) were obtained from the Yale SPORE in Skin Cancer, Yale University, and grown as recommended. SKMEL-103 and M318 cells were provided by Dr. Keiran Smally (Moffitt Cancer Center, Florida). MEL-ST cells were provided by Prof. Robert Weinberg (Whitehead Institute, MIT). All the cell lines were authenticated using STR analysis and tested for mycoplasma regularly using a MycoAlert Mycoplasma detection kit (Lonza, Allendale, NJ). Human IFI6 open reading frame was cloned in pcDNA3.1/hygro and used for rescue experiments. The plasmids pBabe puro-HRAS V12 (plasmid #15269), pBabe puro-HRAS V12 S35 (plasmid# 12274), pBabe puro-MEK-DD (plasmid #15268), and pBabe-puro-BRAFV600E (plasmid #15269) were purchased from Addgene. FG12 was a kind gift of Prof. David Baltimore, and FG12/NRASQ61K was a kind gift of Maria Soengas (CNIO, Spain).
Gupta R., Forloni M., Bisserier M., Dogra S.K., Yang Q, & Wajapeyee N. (2016). Interferon alpha-inducible protein 6 regulates NRASQ61K-induced melanomagenesis and growth. eLife, 5, e16432.
+ Open protocol
+ Expand
5

Establishing Cell Lines with Oncogenic Ras Mutations

Check if the same lab product or an alternative is used in the 5 most similar protocols
To establish cell lines expressing HRasG12V and KRasG12V, GP2-293 packaging cells were co-transfected with pVSV-G (#631530: Clontech, Mountain View, CA, USA) along with either pBABE-puro, pBABE puro H-Ras V12, or pBABE puro K-Ras V12 (#9051, #9052, or #1764: Addgene) using a CalPhos™ Mammalian Transfection Kit (#631312, Clontech) following the manufacturer’s instructions. Retroviral supernatants were used to transduce MCF10A cells in the presence of polybrene (5 mg/mL; Millipore, Burlington, MA, USA). Transduced cells were selected with puromycin (Invivogen, San Diego, CA, USA) for 3 weeks. Selected single cells were isolated, and the expression of HRasG12V and KRasG12V was confirmed by Western blotting.
Lee D.M., Kim I.Y., Lee H.J., Seo M.J., Cho M.Y., Lee H.I., Yoon G., Ji J.H., Park S.S., Jeong S.Y., Choi E.K., Choi Y.H., Yun C.O., Yeo M., Kim E, & Choi K.S. (2024). Akt enhances the vulnerability of cancer cells to VCP/p97 inhibition-mediated paraptosis. Cell Death & Disease, 15(1), 48.
+ Open protocol
+ Expand
6

Plasmid Sources for Cell Transformation

Check if the same lab product or an alternative is used in the 5 most similar protocols
The following plasmids were purchased from AddGene: pWZL hygro H-RasV12 (#18749), pBabe-puro H-RasV12 (#1768), pWZL hygro 12S E1A(#18748), and MSCV-puro-IRES-GFP (#21654). pWZL hygro SV40T-large was a kind gift from Pier Paolo Pandolfi, and 12SLRC H-RasV12/E1A was a kind gift from Marisol Soengas. The MSCV-PIG-FLAG-Inpp4b was cloned by adding FLAG-Inpp4b to the MSCV-puro-IRES-GFP plasmid through Gibson Assembly (NEB).
Mangialardi E.M., Chen K., Salmon B., Vacher J, & Salmena L. (2019). Investigating the duality of Inpp4b function in the cellular transformation of mouse fibroblasts. Oncotarget, 10(59), 6378-6390.
+ Open protocol
+ Expand
7

Oncogenic Ras-Driven Transformation Assays

Check if the same lab product or an alternative is used in the 5 most similar protocols
For monolayer colony-formation assay, 1000 BALB/c fibroblasts were transduced with pCCL-CCR492 and H-RasV12 (Addgene Plasmid 9051: pBABEpuro H-RasV12) expression vectors and plated in 100 mm 2 plates and allowed to grow in appropriate culture medium for 10 days. Fresh media were supplied every 3 days. Colonies were stained with crystal violet dye after formaldehyde fixation.
Sft agar colony-formation assays, 3 x 10 4 cells/ml were transduced in the same conditions and seeded on top of a solidified layer in a volume of 2 ml of 0.5% Bacto Agar (Sigma-Aldrich) over 2 ml 0.4% agar base layers in each six-well plate as previously described [17] (link).
Maldotti M., Incarnato D., Neri F., Krepelova A., Rapelli S., Anselmi F., Parlato C., Basile G., Dettori D., Calogero R, & Oliviero S. (2016). The long intergenic non-coding RNA CCR492 functions as a let-7 competitive endogenous RNA to regulate c-Myc expression. Biochimica et biophysica acta, 1859(10).
+ Open protocol
+ Expand

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

Sign up now

Revolutionizing how scientists
search and build protocols!