prk5 gfp ragb  (Addgene inc)

Journal: bioRxiv

Article Title: SARS-CoV-2 protein ORF3a induces Atg8ylation of lysosomal membranes

doi: 10.1101/2024.09.12.612614

Figure Lengend Snippet: (A,B) High content microscopy (HCM) quantification and image analysis of the effect of Hexa KO on lysotracker intensity. (>500 primary objects examined per well; minimum number of wells, 9). Masks; white: ORF3a-mCherry expressing cells, algorithm-defined cell boundaries; green masks: computer-identified lysotracker dots). p values were determined using GraphPad prism (n=9) ANOVA. Images, a detail from a large database of machine-collected and computer-processed images. Scale bar 10 µm. (C,D) HCM quantification and image analysis of the effect of Hexa KO on ORF3a induced Gal3 puncta formation. (>500 primary objects examined per well; minimum number of wells, 8). Pseudo colours: Red, ORF3a-mCherry; green, Gal3. p values, (n=8) ANOVA. Scale bar 5 µm. (E) HCM analysis of the effect of ORF3a on Gal3 and LAMP1 colocalization in HeLa WT and Hexa KO cells. (>500 primary objects examined per well; minimum number of wells, 6). p values were determined using GraphPad prism (n=6) ANOVA (n=6) ANOVA. (F) Representative micrographs showing the effect of ORF3a expression on Gal3 and LAMP1 colocalization in HeLa WT and Hexa KO cells. Scale bar 5 µm. Pseudo colours: Red, LAMP1; green, Gal3. ORF3a-mCherry is removed from images to avoid confusion. (G,H) HCM analysis of effect of Hexa KO on ORF3a-mCherry induced nuclear translocation of TFEB (>500 primary objects/cells examined per well; minimum number of wells,10). p values were determined using GraphPad prism (n=10) ANOVA. Scale bar 10 µm . (I) Western blot analysis of the effect of ORF3a-mCherry expression on mTOR target p70S6K, in HeLa WT and Hexa KO cells. (J,K) HCM quantifications and micrograph of the effect of ORF3a-mCherry expression on mTOR puncta formation in HeLa WT and Hexa KO cells. Data, means ± SEM, p values were determined using GraphPad prism (n=6) ANOVA; HCM, >500 cells counted per well; minimum number of valid wells 6). Scale bar 5 µm. (L,M) HCM quantifications and micrograph of the effect of ORF3a-mCherry expression on colocalization between mTOR and LAMP1 in HeLa WT and Hexa KO cells. Data, means ± SEM, p values were determined using GraphPad prism (n=6) ANOVA; HCM, >500 cells counted per well; minimum number of valid wells 6. Scale bar 5 µm. Pseudo colours: Red, mTOR; blue, mCherry; green, LAMP1. (N) Co-IP analysis of the effect of ORF3a expression on interaction between FLAG-RagB and endogenous mTOR. (O) Co-IP analysis of the effect of ORF3a expression on interaction between FLAG-TFEB and endogenous mTOR. (P) HCM quantification of the effect of RagB Q99L expression on ORF3a mediated inhibition of lysotracker puncta. Data, means ± SEM, p values were determined using GraphPad prism (n=6) ANOVA; HCM, >500 cells counted per well; minimum number of valid wells 6. (Q) HCM quantification of the effect of RagB Q99L expression on ORF3a mediated formation of Gal3 puncta. Data, means ± SEM, p values were determined using GraphPad prism (n=6) ANOVA; HCM, >500 cells counted per well; minimum number of valid wells 6.

Article Snippet: Plasmids related to SARS-CoV-2 ORF3a-mCherry (#165138), ORF3a-EGFP (#165121), NSP6-mCherry (#165133), NSP4-mCherry (#165132), NSP3-mCherry (#165131), GFP-Gal-3 (#73080), px459 sgFIP200 (#175024), px459 sgAtg5 (#175023), psPAX2 (#12260), pCMV-VSV-G (#8454), pRK5 Flag RagB (#112755), pRK5 GFP RagB Q99L(#112749), and Flag pLJM1 RagB 99L(#19315) were acquired from Addgene.

Techniques: Microscopy, Expressing, Translocation Assay, Western Blot, Co-Immunoprecipitation Assay, Inhibition

Journal: Molecular Cell

Article Title: An mTORC1-to-CDK1 Switch Maintains Autophagy Suppression during Mitosis

doi: 10.1016/j.molcel.2019.10.016

Figure Lengend Snippet:

Article Snippet: pRK5-HA GST RagB Q99L (GTP) , ( ) , Addgene #19303.

Techniques: Biomarker Discovery, Virus, Western Blot, Recombinant, 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

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