Flag rap1b n17

Manufactured by Addgene

Flag-Rap1B-N17 is a plasmid construct that expresses a dominant-negative mutant of the Rap1B protein with a N-terminal Flag epitope tag. Rap1B is a small GTPase that plays a role in various cellular processes. The N17 mutation in Rap1B acts as a dominant-negative, inhibiting the function of endogenous Rap1B.

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Lab products found in correlation

Flag rap1b v12, by Addgene (2 mentions) Gateway lr clonase 2, by Thermo Fisher Scientific (1 mentions) Site directed mutagenesis, by Thermo Fisher Scientific (1 mentions) In fusion hd ecodry cloning kit, by Takara Bio (1 mentions)

2 protocols using flag rap1b n17

Ras Family Modulation in hESCs

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To validate the relevance of the Ras family of small GTPases, human RAP2A cDNA was modified to generate a CA (RAP2A-V12) and DN (RAP2A-N17) isoforms using site-directed mutagenesis (Thermo Fischer). The RAP2A isoforms were then inserted into the lentiviral vector pLenti-PGK Neo or Puro DEST by Gateway LR Clonase II (Thermo Fischer). For RAP1B, CA (bovine Flag-RAP1B-V12, Addgene #118323) and DN (bovine Flag-Rap1B-N17, Addgene #118322) were inserted into the same lentiviral vectors as described above for RAP2A. The resulting vectors were used to transduce WT, PB2 KO, and PB2 OE hESCs.

Junqueira Alves C., Dariolli R., Haydak J., Kang S., Hannah T., Wiener R.J., DeFronzo S., Tejero R., Gusella G.L., Ramakrishnan A., Alves Dias R., Wojcinski A., Kesari S., Shen L., Sobie E.A., Rodrigues Furtado de Mendonça J.P., Azeloglu E.U., Zou H, & Friedel R.H. (2021). Plexin-B2 orchestrates collective stem cell dynamics via actomyosin contractility, cytoskeletal tension and adhesion. Nature Communications, 12, 6019.

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Production and Validation of Rap-related Lentiviruses

Cited 1 time

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pcDNA3 plasmids containing Flag-Cebpa (66978), Flag-Trib1 (131156), Flag-Rap1b N17 (118322), and Flag-Rap1b V12 (118323) were purchased from Addgene. Plasmids containing Rap2a (55666) and Rap2b (55667) were also purchased from Addgene. Plasmids containing Rap2a and Rap2b genes were transferred to the pcDNA3 vector and mutated to dominant=negative N17 and constitutively active V12 using the In-fusion HD EcoDRy Cloning Kit from Takara (639690) according to the manufacturer’s protocol. Furthermore, Rap2a mutants, Cebpa, and Trib1 were cloned into pLVX for lentiviral production. Lentivirus particles were produced in HEK293T LentiX cells (ATCC) as previously described (81 (link)) and used to treat 3T3L1 and PPDIVs.

Valentine J.M., Ahmadian M., Keinan O., Abu-Odeh M., Zhao P., Zhou X., Keller M.P., Gao H., Yu R.T., Liddle C., Downes M., Zhang J., Lusis A.J., Attie A.D., Evans R.M., Rydén M, & Saltiel A.R. (2022). β3-Adrenergic receptor downregulation leads to adipocyte catecholamine resistance in obesity. The Journal of Clinical Investigation, 132(2), e153357.

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