human rab8  (Addgene inc)

Journal: Translational Neurodegeneration

Article Title: Intracellular distribution of amyloid beta peptide and its relationship to the lysosomal system

doi: 10.1186/2047-9158-1-19

Figure Lengend Snippet: Double immunostaining for Aβ 42 (green fluorescence) and different subcellular markers (red fluorescence) in RA differenatiated SH-SY5Y cells. ( A ) Rab8 (Golgi derived vesicles marker), ( B ) Rab9 (trans-Golgi network, Golgi-derived vesicles and late endosome marker), ( C ) LAMP-2 (late endosome and lysosome marker), ( D ) Rab5 (early endosome marker), ( E ) Rab3 (exocytotic vesicle marker) and ( F ) VAMP2 (synaptobrevin, marker for synaptic vesicles) were used as subcellular organelle markers. Scale bar, 5 μm.

Article Snippet: Primary anti-Aβ 1–42 antibodies [ ] (Chemicon, Temecula, CA, USA), and anti-Aβ 1–40 antibodies [ , ] (Chemicon, Temecula, CA, USA), were rabbit polyclonal, while anti-human-Rab8 [ ] (marker for TGN and Golgi-derived secretory vesicles, BD biosciences, Franklin Lakes, NJ, USA), anti-Rab9 [ ] (marker for TGN and late endosomes, Abcam, Cambridge, UK), anti-Rab5 (marker for early endosomes, Pharmingen, San Diego, CA, USA), anti- LAMP-2 (marker for lysosomes and late endosomes, Southern Biotechnology, Birmingham, AL, USA), anti-VAMP 2 (synaptobrevin/VAMP 2, marker for synaptic vesicles, Synaptic Systems, Göttingen, Germany), and anti-Rab3 (marker for synaptic vesicles, Synaptic Systems) antibodies were mouse monoclonal IgG.

Techniques: Double Immunostaining, Fluorescence, Derivative Assay, Marker

Journal: Pharmaceutical Research

Article Title: RAB8 Enhances TMEM205-Mediated Cisplatin Resistance

doi: 10.1007/s11095-011-0562-y

Figure Lengend Snippet: Colocalization of TMEM205 with RAB8. (A) Confocal analysis of the distribution of TMEM205 and TGN-associated proteins in cisplatin-resistant cells. Expression, distribution, and overlapping of TMEM205 (red) and TGN-related proteins (green) is shown for VTI1B, GOLG84, GS28, GM130, NBD-C6-ceramide, and M6PR, respectively. The yellowish color represents colocalization, or a very similar distribution of the two proteins. Scale bar, 20 µm. (B) Images showing colocalization of RAB8 (red) and TMEM205 (green) in the cisplatin-resistant cells, indicating substantial overlapping of these two proteins near the nucleus (blue). Scale bars, 10 µm. (C) Labeling with P230, a TGN marker, shows that there is no co-distribution with TMEM205, and serves as a negative control. Images are representative of three independent experiments. Scale bars, 5 µm.

Article Snippet: Gene Transfection and Assays of Cell Resistance Levels to Cisplatin and Other Compounds Full-length cDNA for the gene encoding human RAB8 was obtained from OriGene (Rockville, MD), then re-inserted into a mammalian expression vector, pcDNA5.1 (Invitrogen, Carlsbad CA), as described by the manufacturer.

Techniques: Expressing, Labeling, Marker, Negative Control

Journal: Pharmaceutical Research

Article Title: RAB8 Enhances TMEM205-Mediated Cisplatin Resistance

doi: 10.1007/s11095-011-0562-y

Figure Lengend Snippet: Overexpression of RAB8. (A) Elevated expression of RAB8 seen in cisplatin-resistant cells. KB-3-1 are the wild-type parental cells; KB-CP.3, KB-CP.5, KB-CP1, and KB-CP20 were selected with cisplatin at concentrations of 0.3, 0.5, 1.0, and 20 µg/ml, respectively. Images shown are representative of three experiments. Scale bars, 10 µm. (B) Effect of cisplatin on expression of RAB8 in KB-3-1 cells. Cells were treated with cisplatin at 0, 1, 3, 10, and 30 mg/ml for 4 hours, or at 0, 0.1, 0.3, 1, 3 µg/ml for 22 hours. The cisplatin-resistant KB-CP.5 cells served as a positive RAB8 expression control.

Article Snippet: Gene Transfection and Assays of Cell Resistance Levels to Cisplatin and Other Compounds Full-length cDNA for the gene encoding human RAB8 was obtained from OriGene (Rockville, MD), then re-inserted into a mammalian expression vector, pcDNA5.1 (Invitrogen, Carlsbad CA), as described by the manufacturer.

Techniques: Over Expression, Expressing, Control

Journal: Pharmaceutical Research

Article Title: RAB8 Enhances TMEM205-Mediated Cisplatin Resistance

doi: 10.1007/s11095-011-0562-y

Figure Lengend Snippet: Dual-transfection of TMEM205 and RAB8. (A) Flow diagram showing the double transfection of RAB8 into KB/M-neor (TMEM205-expressing cells), then selected with the dual selective markers neomycin and hygromycin B for clones carrying both genes; the left column shows a single transfection of RAB8 into the cisplatin-sensitive KB-3-1 cells. (B) Colonies formed after transfection and selection with related reagent(s). KB, without transfection of any vector, serving as a negative control; KB/V, transfected with vector only; KB/R, transfected with RAB8; KB/M+R, transfected with TMEM205 + RAB8. Images shown are representative of three independent experiments. (C) Immunoblotting showing expression levels of RAB8 in individual doubly-transfected clones. KB/V (neo+hyg), which was transfected with a vector carrying both neo and hygromycin markers, but without inserts, served as a negative control. KB-CP.5 served as a positive control for RAB8, and the lower panel shows a Commassie blue-stained gel as a loading control.

Article Snippet: Gene Transfection and Assays of Cell Resistance Levels to Cisplatin and Other Compounds Full-length cDNA for the gene encoding human RAB8 was obtained from OriGene (Rockville, MD), then re-inserted into a mammalian expression vector, pcDNA5.1 (Invitrogen, Carlsbad CA), as described by the manufacturer.

Techniques: Transfection, Expressing, Clone Assay, Selection, Plasmid Preparation, Negative Control, Western Blot, Positive Control, Staining, Control

Journal: Pharmaceutical Research

Article Title: RAB8 Enhances TMEM205-Mediated Cisplatin Resistance

doi: 10.1007/s11095-011-0562-y

Figure Lengend Snippet: Rab8 enhances TMEM205-mediated cisplatin resistance. (A) Confocal images showing expression of TMEM205 and RAB8 in double transfectants (KB/M+R) in comparison to the recipient cells, KB/V. Images are representative of three independent experiments. (B,C) cell survival rates were determined by 3-day CCK8 assays showing levels of resistance of the double transfectants to cisplatin (B), and carboplatin, (C), respectively. The values are means of triplicate determinations. Scale bar, 20 µm.

Article Snippet: Gene Transfection and Assays of Cell Resistance Levels to Cisplatin and Other Compounds Full-length cDNA for the gene encoding human RAB8 was obtained from OriGene (Rockville, MD), then re-inserted into a mammalian expression vector, pcDNA5.1 (Invitrogen, Carlsbad CA), as described by the manufacturer.

Techniques: Expressing, Comparison

Journal: Nanomedicine (London, England)

Article Title: Macrophage endocytic trafficking of antiretroviral nanoparticles

doi: 10.2217/nnm.11.27

Figure Lengend Snippet: Protein markers associated with ritonavir-nanoparticle-containing endosomes.

Article Snippet: Goat antibody (Ab) to Rab11 and Rab7, along with human siRNA to Rab8, Rab11 and Rab14, were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Membrane, Migration, Transduction, Activation Assay

Journal: Nanomedicine (London, England)

Article Title: Macrophage endocytic trafficking of antiretroviral nanoparticles

doi: 10.2217/nnm.11.27

Figure Lengend Snippet: (A–H) Confocal microscopy confirmed distribution of RTV-NP within endocytic compartments. Note that RTV-NP are (A–F) red and (G–H) green, and yellow signifies marker-particle overlap in all panels. (I) Pearson’s colocalization coefficients indicate RTV-NPs are preferentially distributed to Rab11 and Rab14 recycling endosomes compared with early endosomes, Rab8 or Rab7 endosomes, and lysosomes. Analysis of distribution of RTV-NP within acidified (degrading) compartments, identified by pHrodo-dextran beads, revealed minimal overlap indicating RTV-NP likely bypass degradation within the cell and are primarily recycled for release. High RTV-NP colocalization with transferrin also indicates that particles are most likely recycled. Measure bars equal 1 μm. Graphical data represent the mean ± standard error of the mean for n = 3. NP: Nanoparticle; RTV: Ritonavir.

Article Snippet: Goat antibody (Ab) to Rab11 and Rab7, along with human siRNA to Rab8, Rab11 and Rab14, were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Confocal Microscopy, Marker

Journal: Nanomedicine (London, England)

Article Title: Macrophage endocytic trafficking of antiretroviral nanoparticles

doi: 10.2217/nnm.11.27

Figure Lengend Snippet: (A–H) Confocal microscopy confirmed distribution of RTV-NP within endocytic compartments. Note that RTV-NP are (A–F) red and (G–H) green, and yellow signifies marker-particle overlap in all panels. (I) Pearson’s colocalization coefficients indicate RTV-NPs are preferentially distributed to Rab11 and Rab14 recycling endosomes compared with early endosomes, Rab8 or Rab7 endosomes, and lysosomes. Analysis of distribution of RTV-NP within acidified (degrading) compartments, identified by pHrodo-dextran beads, revealed minimal overlap indicating RTV-NP likely bypass degradation within the cell and are primarily recycled for release. High RTV-NP colocalization with transferrin also indicates that particles are most likely recycled. Measure bars equal 1 μm. Graphical data represent the mean ± standard error of the mean for n = 3. NP: Nanoparticle; RTV: Ritonavir.

Article Snippet: Antibodies & reagents Goat antibody (Ab) to Rab11 and Rab7, along with human siRNA to Rab8, Rab11 and Rab14, were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Confocal Microscopy, Marker

Journal: Nanomedicine (London, England)

Article Title: Macrophage endocytic trafficking of antiretroviral nanoparticles

doi: 10.2217/nnm.11.27

Figure Lengend Snippet: (A & B) Disruption of endocytic recycling with siRNA (Rab8, 11 and 14) as well as disruption of cell secretion with brefeldin A resulted in knockout of the associated protein and caused RTV-NPs to be redistributed within monocyte-derived macrophages. (A & B) In each case, siRNA treatment resulted in aggregation of RTV-NPs at the perinuclear region within large vacuoles. (C) siRNA silencing of specific proteins was confirmed by Western blot. High-performance liquid chromatography quantitation of RTV-NP in (D) cells and (E) culture fluids demonstrated that disruption of endocytic recycling and inhibition of secretion significantly increased cellular retention of RTV-NPs and reduced release. Upper p-value signifies difference from control cells and lower p-value signifies difference from cells treated with scrambled siRNA. Measure bars equal 1 μm. Graphical data represent the mean ± standard error of the mean for n = 3. NP: Nanoparticle; RTV: Ritonavir.

Article Snippet: Antibodies & reagents Goat antibody (Ab) to Rab11 and Rab7, along with human siRNA to Rab8, Rab11 and Rab14, were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Disruption, Knock-Out, Derivative Assay, Western Blot, High Performance Liquid Chromatography, Quantitation Assay, Inhibition, Control