All eIF4E, eIF4G (including the Y624A, L629A and L630A binding mutant) and 4EBP1 mutant cDNAs were synthetized and obtained from IDT (Integrated DNA Technologies). eIF4E and eIF4G604–646 were cloned into NanoBit plasmids using the NanoBit PPI starter system (Promega) using XhoI/EcoRI and NheI/EcoRI cloning sites respectively. 4EBP1 mutants were cloned into pCDNA3.1 vector DNA (Thermo Fisher Scientific) harbouring a C-terminal 3× FLAG tag via NheI/BamHI sites to allow mammalian cell overexpression. For bacterial expression, 4EBP1 mutants were cloned into pGEX6P1 using BamHI/EagI cloning sites. GFP and v-Myc coding sequence residing in a pCMV6 mammalian expression vector were obtained from Origene. The bicistronic luciferase reporter construct pcDNA3-rLuc-polIRES-fLuc was purchased from Addgene. PP242, Torin1, Rapamycin, 4EGi-1 and 4E1RCat were purchased from Tocris Bioscience, whilst all other chemicals unless otherwise stated were purchased from Selleck Chemicals. siRNAs targeting either 4EBP1 (ON-TargetPlus Human EI4EBP1, J-003005-12-0005) or non-targeting control (ON-TargetPlus Non-targeting pool, D-001810-10-05) were purchased from DHARMACON.
Pcdna3 rluc polires fluc
Manufactured by Addgene
PcDNA3-rLuc-polIRES-fLuc is a plasmid vector that contains a dual reporter system. It expresses Renilla luciferase (rLuc) and firefly luciferase (fLuc) under the control of a single promoter using an internal ribosome entry site (IRES) sequence.
Automatically generated - may contain errors
Lab products found in correlation
Rapamycin, by Bio-Techne (1 mentions)
Torin1, by Bio-Techne (1 mentions)
Nanobit ppi starter system, by Promega (1 mentions)
Pcmv6 mammalian expression vector, by OriGene (1 mentions)
4e1rcat, by Bio-Techne (1 mentions)
On targetplus non targeting pool d 001810 10 05, by Horizon Discovery (1 mentions)
4egi 1, by Bio-Techne (1 mentions)
Pp242, by Bio-Techne (1 mentions)
Cycloheximide (chx), by Cell Signaling Technology (1 mentions)
Cisplatin, by Selleck Chemicals (1 mentions)
Primary antibody against β actin, by Santa Cruz Biotechnology (1 mentions)
Anti rabbit igg, by Cell Signaling Technology (1 mentions)
Anti mouse igg, by Cell Signaling Technology (1 mentions)
Click it protein reaction buffer kit, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
2 protocols using pcdna3 rluc polires fluc
1
Characterizing eIF4E-eIF4G and 4EBP1 Interactions
2
Isolation and Characterization of NLE from P. nagi
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NLE with a purity of approximately 99.99% was isolated from the seeds of P. nagi as documented in a previous study [6 (link)]. Cycloheximide; anti-rabbit IgG; anti-mouse IgG; and primary antibodies against NRF2, p21, STAT3, ATF4 and GAPDH were obtained from Cell Signaling Technology (Beverly, MA, USA). Primary antibody against β-actin was purchased from Santa Cruz Biotechnology (Dallas, CA, USA). RIPA lysis buffer was procured from Beyotime Biotechnology (Shanghai, China). Cisplatin was purchased from Selleck Chemicals (Houston, TX, USA). The reagents used for the Click-iT assay, Click-iT™ AHA (L-azidohomoalanine), biotin alkyne (PEG4 carboxamide-propargyl biotin), a Click-iT™ protein reaction buffer kit, and anti-streptavidin-HRP, were purchased from Life Technologies (Eugene, OR, USA). The bicistronic reporter plasmid pcDNA3-RLUC-POLIRES-FLUC constructed by Nahum Sonenberg was obtained from Addgene (plasmid # 45642) [15 (link)].
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!