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

Journal: PLoS ONE

Article Title: The Pharmacological Chaperone AT2220 Increases the Specific Activity and Lysosomal Delivery of Mutant Acid Alpha-Glucosidase, and Promotes Glycogen Reduction in a Transgenic Mouse Model of Pompe Disease

doi: 10.1371/journal.pone.0102092

Figure Lengend Snippet: ( A ) GAA and glycogen levels were assessed in tissue lysates prepared from heart and gastrocnemius of 12-week old male Gaa KO, hP545L GAA Tg/KO, and wild-type (denoted as ‘WT’ in the figure) mice. Significant differences in GAA activity and glycogen levels in hP545L GAA Tg/KO and Gaa KO mice compared to wild-type mice are reported as *p<0.05, t-test. Significant differences in GAA activity and glycogen levels in hP545L GAA Tg/KO mice compared to Gaa KO mice are reported as # p<0.05, t-test. Each bar represents the mean ± SEM from four mice per group analyzed in triplicate. Glycogen accumulation ( B ) and lysosomal proliferation (assessed by the quantity of LAMP1) ( C ) were assessed histochemically in Gaa KO, hP545L GAA Tg/KO, and wild-type (denoted as ‘WT’ in the figure) mouse cardiomyocytes and skeletal muscles fibers of the gastrocnemius. Glycogen staining is represented as dark pink spots, and LAMP1 staining as dark brown spots (each denoted with black arrows). The data shown are representative photomicrographs from four mice using a 40× (scale bar: 50 µm) or 20× (scale bar: 100 µm) objective in ( B ) and ( C ), respectively.

Article Snippet: After an overnight incubation with a rat anti-mouse LAMP1 antibody (1∶500 dilution) at 4°C, sections were incubated with components from the Promark rat-on-mouse HRP-polymer kit (Biocare Medical); stain was developed with a Betazoid DAB kit (Biocare Medical).

Techniques: Activity Assay, Muscles, Staining