pegfp rab21 q76l ca  (Addgene inc)

Journal: bioRxiv

Article Title: The Legionella pneumophila type IVb secretion system effector BinA subverts amino acid transport to sensitize TORC1 signaling in macrophages

doi: 10.1101/2025.02.20.639245

Figure Lengend Snippet: Kinetics of TORC1 activation triggered by starvation/refeeding stimulation with 500µM Arg in HK293 cells producing BinA in the presence or absence of different Rab5 (A-B) , Rab21 (C-D) , or Rab22a (E-F) alleles. (B, D and F) Quantitative analyses of band signal intensities show Averages ± StDev from at least three biological repeats (left panels) and the respective area-under-the curve (AUC) analyses are presented in the right panels. Statistical analyses were completed with one- way ANOVA with Dunnett’s multiple comparison test using the ‘BinA+GFP’ as control group and p-values are indicated in the respective data panels.

Article Snippet: The murine Rab21, Rab21 Q76L and Rab21 T31N alleles were a gift from Johanna Ivaska (Addgene plasmids # 83421, 83422, 83423; RRID:Addgene_83421, RRID:Addgene_83422, RRID:Addgene_83423) [ ] .

Techniques: Activation Assay, Comparison, Control

Journal: bioRxiv

Article Title: The Legionella pneumophila type IVb secretion system effector BinA subverts amino acid transport to sensitize TORC1 signaling in macrophages

doi: 10.1101/2025.02.20.639245

Figure Lengend Snippet: Kinetics of TORC1 activation triggered by starvation/refeeding stimulation with either 500µM Arg (A) or 100 µM Leu (B) in HK293 cells producing RagA Q66L in the presence or absence of different GDP-locked Rab5, Rab21 or Rab22a alleles. (A-B) Immunoblots from representative experiments are shown in the left panels and AUC analyses from three biological repeats are graphed in the right panels. Statistical analyses were completed with one-way ANOVA with Dunnett’s multiple comparison test using the ‘GFP-RagA Q66L’ as control group and p-values are indicated in the respective data panels.

Article Snippet: The murine Rab21, Rab21 Q76L and Rab21 T31N alleles were a gift from Johanna Ivaska (Addgene plasmids # 83421, 83422, 83423; RRID:Addgene_83421, RRID:Addgene_83422, RRID:Addgene_83423) [ ] .

Techniques: Activation Assay, Western Blot, Comparison, Control

Journal: bioRxiv

Article Title: The Legionella pneumophila type IVb secretion system effector BinA subverts amino acid transport to sensitize TORC1 signaling in macrophages

doi: 10.1101/2025.02.20.639245

Figure Lengend Snippet: Kinetics of TORC1 activation triggered by starvation/refeeding stimulation with 1mM Arg in HK293 cells producing GFP or different Rab5 (A) , Rab21 (B) , or Rab22a (C) alleles. Band signal intensity in the phospho-immunoblot for rS6p for each condition was quantified and is presented below the respective figure panel as fold change from untreated cells. The data shown is from one experiment out of three biological replicates.

Article Snippet: The murine Rab21, Rab21 Q76L and Rab21 T31N alleles were a gift from Johanna Ivaska (Addgene plasmids # 83421, 83422, 83423; RRID:Addgene_83421, RRID:Addgene_83422, RRID:Addgene_83423) [ ] .

Techniques: Activation Assay, Western Blot

Journal: bioRxiv

Article Title: The Legionella pneumophila type IVb secretion system effector BinA subverts amino acid transport to sensitize TORC1 signaling in macrophages

doi: 10.1101/2025.02.20.639245

Figure Lengend Snippet: (A) Early translocation of multiple T4bSS effectors causes (i) inhibition of the host translation machinery resulting in amino acids accumulation, which in turn triggers TORC1 signaling at approximately 2 hours post bacterial internalization. Enhancement of amino acid import driven by BinA (ii) sustains TORC1 signaling for several hours and represents an additional regulatory layer. (B) At least two distinct amino acid pools are sourced independently to provide sensory input to TORC1. One input likely originates from an organelle controlled by the small GTPases Rab21 and Rab22 such as the recycling endosomal compartment (REC). The second is regulated the Rab5 and thus is likely further down the endocytic pathway presumably of endosome/lysosome origin (E/LY). In cells containing BinA, TORC1 is sensitized through increased amino acid import from the Rab21/Rab22 compartment in a manner that also renders the Rab5-dependent pathway superfluous potentially through a switch in the utilization of transporters and/or amino acid sensors. Ectopic expression of RagA Q66L mutant elicits a preference for the Rab5- dependent amino acid pool. Both pools autonomously can trigger TORC1 signaling in the absence of BinA and RagA Q66L, which ensures TORC1 response capacity is retained irrespective of endocytic trafficking.

Article Snippet: The murine Rab21, Rab21 Q76L and Rab21 T31N alleles were a gift from Johanna Ivaska (Addgene plasmids # 83421, 83422, 83423; RRID:Addgene_83421, RRID:Addgene_83422, RRID:Addgene_83423) [ ] .

Techniques: Translocation Assay, Inhibition, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: Identification of a new, Rab14-dependent, endo-lysosomal pathway

doi: 10.1101/2021.03.25.436964

Figure Lengend Snippet: (A) Colocalization quantitation between the indicated endocytosed material and the indicated GFP-labeled Rab proteins ectopically expressed in HeLa KCNN4 knockout cells. The results correspond to mean ± SD of three independent experiments. (B) Colocalization quantitation between the indicated endocytosed material and EEA1 in cells transfected with Rab14, Rab21 or Rab22 dominant negative constructs. The data correspond to the mean ± SD of three independent experiments. Statistical analysis was performed using ANOVA test with Dunett’s correction, based on AUC values. The p-values correspond to the comparison between the cells within the same population transfected or not with the dominant negative constructs.

Article Snippet: GFP-hRab21(T31N) (#1039) plasmid encoding GFP-labeled version of dominant negative Rab21 mutant was from Addgene (Cat# 83423).

Techniques: Quantitation Assay, Labeling, Knock-Out, Transfection, Dominant Negative Mutation, Construct, Comparison