eif4e full length coding sequence  (Addgene inc)

Journal: The Journal of Biological Chemistry

Article Title: The PP242 Mammalian Target of Rapamycin (mTOR) Inhibitor Activates Extracellular Signal-regulated Kinase (ERK) in Multiple Myeloma Cells via a Target of Rapamycin Complex 1 (TORC1)/ Eukaryotic Translation Initiation Factor 4E (eIF-4E)/RAF Pathway and Activation Is a Mechanism of Resistance *

doi: 10.1074/jbc.M111.304626

Figure Lengend Snippet: Effects on 4E-BP1/eIF-4E mediate ERK activation. In A, 8226 or MM1.S cells treated with IGF-1 (200 ng/ml) ± rapamycin or pp242 for 30 min followed by eIF-4E precipitation and subsequent immunoblot of the precipitate for eIF-4E, 4E-BP1, and eIF-4G presence. In B, similar experiment performed in 8226 cells without presence of IGF-1. In C, empty vector (EV) or HA-eIF-4E- stably transfected MM1.S cells (left panel) or 8226 cells (right panel) treated with increasing concentrations of pp242 followed by immunoblot assay for phospho-ERK, total ERK, and HA tag. In D, empty vector or HA-eIF-4E transiently transfected 8226 cells treated with increasing concentrations of pp242 followed by immunoblot assay for phosphorylated and total ERK or phosphorylated and total MEK. In E, empty vector or eIF-4E-transfected cells treated with increasing concentrations of pp242 followed by immunoprecipitation of c-RAF and assay for raf in vitro kinase activity against MEK substrate. Fold increase in kinase activity calculated as described under “Experimental Procedures.”

Article Snippet: The HA-tagged eif4e full-length coding sequence was isolated from pHA-eif4e (obtained from Addgene, plasmid 17343) by PCR, using primers Spe1HA-eif4e (5-AAAGACTAGTATGTACCCATACGACGTCCC-3) and Xho1HA-eif4e (5-GATGCATGCTCGAGTTAAACA-3) to produce 5′-Spe1HA-eif4eXho1–3′ PCR product.

Techniques: Activation Assay, Western Blot, Plasmid Preparation, Stable Transfection, Transfection, Immunoprecipitation, In Vitro, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: The PP242 Mammalian Target of Rapamycin (mTOR) Inhibitor Activates Extracellular Signal-regulated Kinase (ERK) in Multiple Myeloma Cells via a Target of Rapamycin Complex 1 (TORC1)/ Eukaryotic Translation Initiation Factor 4E (eIF-4E)/RAF Pathway and Activation Is a Mechanism of Resistance

doi: 10.1074/jbc.m111.304626

Figure Lengend Snippet: FIGURE 6. Effects on 4E-BP1/eIF-4E mediate ERK activation. In A, 8226 or MM1.S cells treated with IGF-1 (200 ng/ml) rapamycin or pp242 for 30 min followed by eIF-4E precipitation and subsequent immunoblot of the precipitate for eIF-4E, 4E-BP1, and eIF-4G presence. In B, similar experiment performed in 8226 cells without presence of IGF-1. In C, empty vector (EV) or HA-eIF-4E- stably transfected MM1.S cells (left panel) or 8226 cells (right panel) treated with increasing concentrations of pp242 followed by immunoblot assay for phospho-ERK, total ERK, and HA tag. In D, empty vector or HA-eIF-4E transiently transfected8226cellstreatedwithincreasingconcentrationsofpp242followedbyimmunoblotassayforphosphorylatedandtotalERKorphosphorylatedand total MEK. In E, empty vector or eIF-4E-transfected cells treated with increasing concentrations of pp242 followed by immunoprecipitation of c-RAF and assay for raf in vitro kinase activity against MEK substrate. Fold increase in kinase activity calculated as described under “Experimental Procedures.”

Article Snippet: The HA-tagged eif4e full-length coding sequence was isolated from pHA-eif4e (obtained from Addgene, plasmid 17343) by PCR, using primers Spe1HA-eif4e (5-AAAGACTAGTATGTACCCATACGACGTCCC-3) and Xho1HA-eif4e (5-GATGCATGCTCGAGTTAAACA-3) to produce 5 -Spe1HA-eif4eXho1–3 PCR product.

Techniques: Activation Assay, Western Blot, Plasmid Preparation, Stable Transfection, Transfection, Immunoprecipitation, In Vitro, Activity Assay