Journal: Traffic (Copenhagen, Denmark)

Article Title: Toxin Pores Endocytosed During Plasma Membrane Repair Traffic into the Lumen of MVBs for Degradation

doi: 10.1111/j.1600-0854.2011.01323.x

Figure Lengend Snippet: Cells were treated with 250 ng/mL GFP-SLO at 4°C for 5 min, washed and shifted to 37°C in the presence of Ca2+ for increasing periods of time. Samples were then fixed and processed for cryo-immuno EM and stained with anti-GFP and/or anti-Lamp1 antibodies. A) Cryo-immuno EM localization of GFP-SLO (arrows) at the plasma membrane immediately after permeabilization (0 min) and on increasingly larger endocytic vesicles over time (1–30 min). Bar = 100 nm. B) The percentage of the total SLO detected at the plasma membrane decreases from ~ 60% at 0 min to about 12% as early as 5 min after cell permeabilization, while a corresponding increase is seen in intracellular SLO. C) The distance (nm) of intracellular SLO to the plasma membrane increases progressively over time. Quantifications were performed in 10–17 cells containing 175–500 gold particles. Results are expressed as the mean ± SEM. D) Representative cryo-immuno EM images of GFP-SLO (10-nm gold) and Lamp1 (5-nm gold) staining in cells treated with GFP-SLO for 30 min. GFP-SLO and Lamp1 staining colocalize on endosomal structures containing intraluminal vesicles at 30 min after cell permeabilization and repair. Bar = 100 nm.

Article Snippet: Immunoblot and immuno-EM assays were performed using rabbit anti-GFP to detect GFP-SLO (Invitrogen), rabbit anti-ubiquitin (Abcam), rabbit anti Vps24 (Santa Cruz Biotechnology), mouse anti-actin (Sigma) and mouse anti-Lamp1 monoclonal antibodies (mAb) (LYIC6; provided by I. Mellman, Genentech).

Techniques: Staining