Journal:

Article Title: Rab6 Regulates Both ZW10/RINT-1- and Conserved Oligomeric Golgi Complex-dependent Golgi Trafficking and Homeostasis

doi: 10.1091/mbc.E07-01-0080

Figure Lengend Snippet: HeLa cells double-treated with COG3 and ZW10 siRNAs display a phenotype that shows vesicular accumulation of GlcNAcT1 as observed with COG3 siRNA treatment. HeLa cells stably expressing GlcNAcT1-myc (red channel) were mock (control, A) or COG3 (B). ZW10 (C) and double (ZW10 + COG3) (D) siRNA treated for 72 h, 100 nM individual duplex siRNA concentration, fixed, and stained with anti-myc antibody, anti-GM130 (green channel); and DNA (DAPI, blue channel). Image stacks were taken with the CARV II spinning disk confocal accessory and projected into a single XY plane.

Article Snippet: Widefield microscopy and imaging were performed with a Zeiss Axiovert 200M microscope (Carl Zeiss, Jena, Germany) fitted with high numerical aperture (0.80 or 1.4) 20, 63, and 100× objectives and a CARV II spinning disk confocal accessory (BD Bioimaging, Rockville, MD) mounted to the sideport of the microscope.

Techniques: Stable Transfection, Expressing, Concentration Assay, Staining

Journal:

Article Title: Rab6 Regulates Both ZW10/RINT-1- and Conserved Oligomeric Golgi Complex-dependent Golgi Trafficking and Homeostasis

doi: 10.1091/mbc.E07-01-0080

Figure Lengend Snippet: Rab6 depletion inhibits COG3 siRNA-induced fragmentation of Golgi and accumulation of GlcNAcT1 in vesicles. (A–D) Cells double-treated with Rab6 + COG3 siRNAs show compact Golgi phenotype. HeLa cells stably expressing GlcNAcT1-myc were mock (control) (A) or COG3 (B), Rab6 (C), and double (Rab6 + COG3) (D) siRNA treated for 72 h, fixed, and stained with anti-myc antibody. Myc signal was collected as confocal image stacks by using a CARV II confocal accessory. (E) Total cell lysates from A to D were immunoblotted with anti-Cog3 (top row), anti-Rab 6 (middle row), and anti-GAPDH (bottom row) antibodies. (F–H) Rab5 siRNA knockdown does not interfere with COG3 siRNA-induced fragmentation of Golgi. HeLa cells stably expressing GalNAcT2-GFP were COG3 (F) or double (Rab5/COG3) (G) siRNA treated. (H) Total cell lysates from F and G were immunoblotted with anti-Rab5 and anti-β-actin as a loading control. Efficiency of knockdown was ∼90%. Widefield images. Arrows indicate fragmented Golgi apparatus. Bar, 10 μm.

Article Snippet: Widefield microscopy and imaging were performed with a Zeiss Axiovert 200M microscope (Carl Zeiss, Jena, Germany) fitted with high numerical aperture (0.80 or 1.4) 20, 63, and 100× objectives and a CARV II spinning disk confocal accessory (BD Bioimaging, Rockville, MD) mounted to the sideport of the microscope.

Techniques: Stable Transfection, Expressing, Staining

Journal:

Article Title: Rab6 Regulates Both ZW10/RINT-1- and Conserved Oligomeric Golgi Complex-dependent Golgi Trafficking and Homeostasis

doi: 10.1091/mbc.E07-01-0080

Figure Lengend Snippet: Rab6 knockdown reduces Golgi fragmentation caused by microinjection of Cog3 antibody. HeLa cells stably expressing GalNAcT2-GFP were mock (A and B) or Rab6 (C and D) siRNA treated for 72 h. Cells were microinjected with affinity-purified anti-Cog3 (A and C) or preimmune (B and D) antibodies and fixed 4 h after microinjection. GFP signal was collected as confocal image stacks by using a CARV II confocal accessory. Arrow, fragmented Golgi; arrowhead, compact Golgi; asterisk, injected cells. Bar, 10 μm.

Article Snippet: Widefield microscopy and imaging were performed with a Zeiss Axiovert 200M microscope (Carl Zeiss, Jena, Germany) fitted with high numerical aperture (0.80 or 1.4) 20, 63, and 100× objectives and a CARV II spinning disk confocal accessory (BD Bioimaging, Rockville, MD) mounted to the sideport of the microscope.

Techniques: Stable Transfection, Expressing, Affinity Purification, Injection