rragc cdna  (BioChain Institute)

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Recurrent mutations in the MTOR regulator RRAGC in follicular lymphoma

doi: 10.1158/1078-0432.CCR-16-0609

Figure Lengend Snippet: FL-associated RRAGC mutations cluster on the protein surface surrounding the GTP/GDP binding site. Upper: Location of identified RRAGC missense mutations in a linear schema of RRAGC. Lower: Location of identified RRAGC missense mutations in the crystal complex (PDB:3LLU) of the RRAGC nucleotide binding domain (G-domain) bound with the nucleotide analog phosphoaminophosphonic acid guanylate ester (GNP), shown in stick representation. Amino acid residues undergoing mutation are labeled and shown in stick representation, while other residues within 4 angstroms of GNP are shown in line representation. A magnesium ion is shown as a sphere. The figure was generated with PyMOL.

Article Snippet: Reagents and mutagenesis A pCMV-SPORT plasmid containing the RRAGC cDNA (cat#: MHS6278-202757712; accession: {"type":"entrez-nucleotide","attrs":{"text":"BC016668","term_id":"16741747","term_text":"BC016668"}} BC016668 ) was purchased from ThermoScientific, and used as a template to generate mutant RRAGC cDNAs using the QuikChange® Lightning Site-Directed Mutagenesis Kit (Stratagene/Agilent, La Jolla, CA).

Techniques: Binding Assay, Mutagenesis, Labeling, Generated

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Recurrent mutations in the MTOR regulator RRAGC in follicular lymphoma

doi: 10.1158/1078-0432.CCR-16-0609

Figure Lengend Snippet: Details of gene mutations in FL cases carrying RRAGC mutations.

Article Snippet: Reagents and mutagenesis A pCMV-SPORT plasmid containing the RRAGC cDNA (cat#: MHS6278-202757712; accession: {"type":"entrez-nucleotide","attrs":{"text":"BC016668","term_id":"16741747","term_text":"BC016668"}} BC016668 ) was purchased from ThermoScientific, and used as a template to generate mutant RRAGC cDNAs using the QuikChange® Lightning Site-Directed Mutagenesis Kit (Stratagene/Agilent, La Jolla, CA).

Techniques:

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Recurrent mutations in the MTOR regulator RRAGC in follicular lymphoma

doi: 10.1158/1078-0432.CCR-16-0609

Figure Lengend Snippet: FL-associated RRAGC mutations are intrinsically mTOR activating. Upper: HEK293T cells stably retrovirally infected expressing HA-tagged RRAGC wild type or various RRAGC mutants were grown in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS; +) or alternatively for the last 1 h of culture in RPMI1640 medium that was free of the amino acid leucine and supplemented with dialyzed 10% FBS (−). Cells were harvested and prepared for immunoblotting with various antibodies as indicated. Short and long exposures for RPS6KB/S6K (S6K) and p-Thr389-RPS6KB/S6K (p-S6K) are shown. CHO-IR + INS: Insulin receptor transfected CHO cells stimulated with insulin (immunoblot controls). Measurements of HA-RRAGC expression levels were aided by the slower migration of the HA-tagged RRAGC WT and mutant proteins in SDS-PAGE resulting in a doublet band. Lower: Displayed are combined quantitation results (p-S6K/total S6K) from 3 independent experiments using ImageJ densitometry results indexed to the measurements for RRAGC wt.

Article Snippet: Reagents and mutagenesis A pCMV-SPORT plasmid containing the RRAGC cDNA (cat#: MHS6278-202757712; accession: {"type":"entrez-nucleotide","attrs":{"text":"BC016668","term_id":"16741747","term_text":"BC016668"}} BC016668 ) was purchased from ThermoScientific, and used as a template to generate mutant RRAGC cDNAs using the QuikChange® Lightning Site-Directed Mutagenesis Kit (Stratagene/Agilent, La Jolla, CA).

Techniques: Stable Transfection, Infection, Expressing, Western Blot, Transfection, Migration, Mutagenesis, SDS Page, Quantitation Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Recurrent mutations in the MTOR regulator RRAGC in follicular lymphoma

doi: 10.1158/1078-0432.CCR-16-0609

Figure Lengend Snippet: Measurements of p-Thr389-RPS6KB/S6K levels in RRAGC WT or mutant stably lentivirally transduced lymphoma cell lines. Upper: Lymphoma cell lines stably lentivirally infected with constructs expressing HA-tagged RRAGC wild type or various RRAGC mutants as indicated were grown in RPMI or DMEM medium supplemented with 10% FBS (+) or alternatively for the last 1 h of culture in medium that was free of the amino acid leucine and supplemented with dialyzed 10% FBS (−). Cells were harvested and prepared for immunoblotting with various antibodies as indicated. Short and long exposures for p-Thr389-RPS6KB/S6K are shown. CHO-IR + INS: Insulin receptor transfected CHO cells stimulated with insulin (immunoblot controls). Measurements of HA-RRAGC expression levels were aided by the slower migration of the HA-tagged RRAGC WT and mutant proteins in SDS-PAGE resulting in a doublet band. Lower: Displayed are combined quantitation results (p-S6K/total S6K) from 3 independent experiments per cell line using ImageJ densitometry results indexed to the measurements for RRAGC wt.

Article Snippet: Reagents and mutagenesis A pCMV-SPORT plasmid containing the RRAGC cDNA (cat#: MHS6278-202757712; accession: {"type":"entrez-nucleotide","attrs":{"text":"BC016668","term_id":"16741747","term_text":"BC016668"}} BC016668 ) was purchased from ThermoScientific, and used as a template to generate mutant RRAGC cDNAs using the QuikChange® Lightning Site-Directed Mutagenesis Kit (Stratagene/Agilent, La Jolla, CA).

Techniques: Mutagenesis, Stable Transfection, Infection, Construct, Expressing, Western Blot, Transfection, Migration, SDS Page, Quantitation Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Recurrent mutations in the MTOR regulator RRAGC in follicular lymphoma

doi: 10.1158/1078-0432.CCR-16-0609

Figure Lengend Snippet: Analysis of TOR activation resulting from Gtr2 mutations in yeast. (A) Alignment of human and yeast RRAGC and Gtr2 proteins, respectively. Mutated residues are boxed. (B) Yeast cells with the PHO8 locus replaced with pho8∆60 were chromosomally-tagged with Gtr2-3HA WT and the indicated mutants. Protein extracts were prepared from cells in growing conditions (0 h) or after 2 h of nitrogen starvation, and assayed for Pho8∆60-dependent phosphatase activity.

Article Snippet: Reagents and mutagenesis A pCMV-SPORT plasmid containing the RRAGC cDNA (cat#: MHS6278-202757712; accession: {"type":"entrez-nucleotide","attrs":{"text":"BC016668","term_id":"16741747","term_text":"BC016668"}} BC016668 ) was purchased from ThermoScientific, and used as a template to generate mutant RRAGC cDNAs using the QuikChange® Lightning Site-Directed Mutagenesis Kit (Stratagene/Agilent, La Jolla, CA).

Techniques: Activation Assay, Activity Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Recurrent mutations in the MTOR regulator RRAGC in follicular lymphoma

doi: 10.1158/1078-0432.CCR-16-0609

Figure Lengend Snippet: A RRAGC mutants demonstrate increased HA-RPTOR binding in co-immunoprecipitation studies in transiently transfected HEK293T cells. Upper: HEK293T cell were transiently co-transfected as indicated with expression plasmids coding for HA-RPTOR, RRAGB, RRAGC WT or the indicated RRAGC mutants. CHAPS detergent lysates were prepared and subjected to anti-HA-bead conjugate-mediated immunoprecipitations. Bound protein was eluted and fractioned by SDS-PAGE and prepared for immunoblotting with the indicated antibodies. Leucine starvation (Leu −) was performed in parallel transfections. Lysate: aliquots of lysates prior to immunoprecipitation. Lower: Displayed are combined quantitation results (RRAGC IP band intensities divided by RRAGC detergent cell lysates band intensities normalized to raptor IP band densities) from 3 independent experiments using ImageJ densitometry results indexed to the measurements for RRAGC wt.

Article Snippet: Reagents and mutagenesis A pCMV-SPORT plasmid containing the RRAGC cDNA (cat#: MHS6278-202757712; accession: {"type":"entrez-nucleotide","attrs":{"text":"BC016668","term_id":"16741747","term_text":"BC016668"}} BC016668 ) was purchased from ThermoScientific, and used as a template to generate mutant RRAGC cDNAs using the QuikChange® Lightning Site-Directed Mutagenesis Kit (Stratagene/Agilent, La Jolla, CA).

Techniques: Binding Assay, Immunoprecipitation, Transfection, Expressing, SDS Page, Western Blot, Quantitation Assay