Journal: Cell metabolism

Article Title: Arginase 2 Suppresses Renal Carcinoma Progression via Biosynthetic Cofactor Pyridoxal Phosphate Depletion and Increased Polyamine Toxicity

doi: 10.1016/j.cmet.2018.04.009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The ARG2 (H160F) mutant was generated using Stratagene’s QuikChange II mutagenesis kit (Agilent). pLJM1-Flag-RagB GTP -Q99L mutant plasmid was obtained from Addgene (plasmid no. 19315) and subcloned into pCDH-CMV-MCS-Neomycin (System Biosciences).

Techniques: Recombinant, Arginase Activity Assay, Colorimetric Assay, Mutagenesis, Software

Journal: Molecular cell

Article Title: Translational control through differential ribosome pausing during amino acid limitation in mammalian cells

doi: 10.1016/j.molcel.2018.06.041

Figure Lengend Snippet: (A) Representative western blots for phosphorylated and total S6K in HEK293T cells after growth in rich medium or limitation for leucine or arginine for 3 hours, with or without (n.t.) 250 nM Torin1. Bar graph shows percent of protein that is phosphorylated, relative to the maximum; error bars represent the standard error of the mean from three technical replicates. (B) Changes in codon-specific ribosome density in the hrGFP cell line (as shown in C) after 3 hours of leucine or arginine limitation with 250 nM Torin1, relative to rich medium. (C) Representative western blots for phosphorylated S6K, total S6K, and FLAG after growth in rich medium, or 3 hours of leucine or arginine limitation in HEK293T cells stably expressing either hrGFP, FLAG-RagB-WT (RagB-WT), or FLAG-RagB-Q99L (RagB-Q99L). Bar graph shows percent of protein that is phosphorylated, relative to the maximum in the RagB-Q99L cell line; error bars represent the standard error of the mean from three technical replicates. (D,E) Representative western blots for phosphorylated and total eIF2α (D) or S6K (E) after growth in rich medium, or 3 hours of leucine or arginine limitation in the HEK293T (WT) or GCN2 KO cell lines. Bar graphs show percent of protein that is phosphorylated, relative to the maximum in WT cells; error bars represent the standard error of the mean from three technical replicates. (F) Changes in codon-specific ribosome density for WT, hrGFP, FLAG-RagB-Q99L, and GCN2 KO cell lines following 6 hours of limitation for leucine or arginine, relative to rich medium.

Article Snippet: We cloned sequences for FLAG-RagB-WT and FLAG-RagB-Q99L into this plasmid in place of hrGFP, from sequences in FLAG pLJM1 RagB wt (Addgene # 19313) and FLAG pLJM1 RagB 99L (Addgene # 19315) from David Sabatini ( Sancak et al., 2008 ).

Techniques: Western Blot, Stable Transfection, Expressing

Journal: Molecular cell

Article Title: Translational control through differential ribosome pausing during amino acid limitation in mammalian cells

doi: 10.1016/j.molcel.2018.06.041

Figure Lengend Snippet: KEY RESOURCES TABLE :

Article Snippet: We cloned sequences for FLAG-RagB-WT and FLAG-RagB-Q99L into this plasmid in place of hrGFP, from sequences in FLAG pLJM1 RagB wt (Addgene # 19313) and FLAG pLJM1 RagB 99L (Addgene # 19315) from David Sabatini ( Sancak et al., 2008 ).

Techniques: Recombinant, Staining, Western Blot, Stripping, Hybridization, SYBR Green Assay, Northern Blot, Homologous Recombination, Clone Assay, Sequencing, FLAG-tag, Software