Journal: Scientific Reports

Article Title: Rab6 is required for rapid, cisternal-specific, intra-Golgi cargo transport

doi: 10.1038/s41598-020-73276-w

Figure Lengend Snippet: Rab6 knockdown strongly inhibits intra-Golgi cargo transport. Confocal immunofluorescence, orthogonal views of tsO45G-GFP distribution in the Golgi apparatus of HeLa cells stained for p115 (cis-Golgi, red) and TGN46 (TGN, blue) at various times. ( A ) 0 time, 20 min chase and 40 min chase. At 0 time, there is little, if any correspondence between tsO45G-GFP distribution and a Golgi marker while at 20 min there is extensive correspondence in either Control or Rab6 knockdown cells, and at 40 min chase, Control cells exhibit little to no correspondence in TGN46 distribution and tsO45-GFP. In Rab6 knockdown cells, most of the tsO45G-GFP corresponded in distribution to TGN46 (blue). ( B ) Immunoblot demonstrating the high level of Rab6 protein knockdown with siRNA treatment. ( C ) Immunofluorescence evidence for high level Ra6 protein knockdown across the HeLa cell population. Software used includes iVision 4.5 ( https://www.biovis.com/ ), Huygens Professional 4.3.0 p3 ( https://www.svi.nl ) and Photoshop 2019 ( https://adobe.com ).

Article Snippet: Transfections were carried out as previously described , however on Day 4 after incubations with either siControl or siRab6 A/A′, HeLa cells were transfected with plasmids encoding GFP-tagged tsO45 mutant VSVG protein using FuGENE HD transfection reagent (Promega).

Techniques: Immunofluorescence, Staining, Marker, Western Blot, Software

Journal:

Article Title: Rab8 Regulates Basolateral Secretory, But Not Recycling, Traffic at the Recycling Endosome

doi: 10.1091/mbc.E07-09-0902

Figure Lengend Snippet: Ablation of the RE causes mis-sorting of newly synthesized VSV-G. All panels show MDCKT cells (4 d after plating) that have internalized Tfn-HRP into the RE. (A, C, E, G, I, and K) Control cells treated with DAB but not H2O2; (B, D, F, H, J, and L) cells treated with DAB and H2O2. (A and B) Basolaterally applied Alexa-546 Tfn internalized into MDCKT cells for 8 min labels early (small arrow) and recycling (large arrow) endosomes. REs are not visible in RE-ablated cells. (C and D) Basolaterally applied Alexa-546 Tfn internalized into control MDCKT cells for 25 min labels RE (large arrows). Small peripheral early endosomes are visible in RE-ablated cells (small arrows). (E and F) In MDCKT cells labeled for Gp-114, both control and RE-ablated cells are labeled apically. (G and H) In MDCKT cells labeled for p58, both control and RE-ablated cells are labeled basolaterally. (I and J) MDCKT cells expressing VSV-G tsO45 released at the permissive temperature for 2 h after RE ablation. Green arrow in I indicates normal basolateral distribution of VSV-G. Red arrow in J indicates putative apical VSV-G in RE-ablated cells. (K and L) MDCKT cells as in I and J containing cellular GFP-VSV-G (green) and with surface VSV-G labeled by anti-VSV-G ectodomain antibody (red). Red arrow indicates mis-sorted apical VSV-G in ablated cells. Bars, 10 μm.

Article Snippet: We are especially indebted to Agnes Ang, and Ira Mellman (both of Genentech) for providing the dsRFP-Rab8 constructs as well as the YFP-VSV-G tsO45 mutant construct.

Techniques: Synthesized, Control, Labeling, Expressing