rab8 constructs  (Addgene inc)

Journal: Human Molecular Genetics

Article Title: LRRK2 phosphorylates membrane-bound Rabs and is activated by GTP-bound Rab7L1 to promote recruitment to the trans-Golgi network

doi: 10.1093/hmg/ddx410

Figure Lengend Snippet: LRRK2 phosphorylation of GTP-bound Rab10 may decrease interaction with the GAP protein AS160. (A) Structural alignment of Rab8-GTP (Green) and Rab8-GDP (magenta). The unstructured switch II motif in the GDP-bound form is shown as dashed line. The Thr72 side chain is shown as sticks. (B) Assessment of purity of recombinant proteins isolated from HEK293 cells, for G2019S-LRRK2, WT-Rab10, and WT-Rab7L1, by SDS-PAGE followed by coomassie staining. All proteins showed >95% purity. (C) Calculated ratios of pRab10 from in vitro kinase assays in the presence of 500 μM of GTP, or GDP, with MLi2 (100 nM), as indicated. (D) Lysates from HEK293 cells co-transfected with LRRK2 and WT, TA, TD-Rab10, as indicated. MLi2 was included at 100 nM as indicated. Representative phos-tag analysis and immunoblots are shown. (E) Live cell imaging of SH-SY5Y cells. Wide-field fluorescence of the eGFP-tagged Rab10 construct is shown on a dark phase-contrast background, 16 h after transfection. (F) Cells over-expressing the Rab10 GAP AS160 or mock (empty vector) controls were lysed and incubated with bead-immobilized WT, TA or TD-Rab10. Complexes were analysed by immunoblots as shown and quantified (G) as column graphs depicting Rab10 interaction with Rab10 variants, relative to WT-Rab10. All data are averaged from three independent experiments or are representative of three independent experiments. Data are means ± SEM; significance assessed by one-way ANOVA with Tukey’s post hoc test. *P < 0.01, **P < 0.001.

Article Snippet: Rab8 constructs are obtained from Addgene (Plasmids 86075, 86076, 86077) courtesy of the Lei Lu laboratory. pEGFP-Rab7L1 plasmids are generated as previously described ( 9 ).

Techniques: Phospho-proteomics, Recombinant, Isolation, SDS Page, Staining, In Vitro, Transfection, Western Blot, Live Cell Imaging, Fluorescence, Construct, Expressing, Plasmid Preparation, Incubation

Journal: eLife

Article Title: Golgi-independent secretory trafficking through recycling endosomes in neuronal dendrites and spines

doi: 10.7554/eLife.27362

Figure Lengend Snippet:

Article Snippet: transfected construct (Rab8/Rab8DN) , GFP-Rab8 , , Derived from Addgene (24898, 24899) , Gift from Dr. Maxence Nachury.

Techniques: Transfection, Construct, Generated, Derivative Assay, Plasmid Preparation, Western Blot, Recombinant