lfn gfp  (Thermo Fisher)

Journal:

Article Title: A fragment of anthrax lethal factor delivers proteins to the cytosol without requiring protective antigen

doi: 10.1073/pnas.1131930100

Figure Lengend Snippet: HeLa cells internalize LFn-GFP in the absence of PA, but do not internalize GFP. HeLa cells were incubated with Texas red-conjugated transferrin and GFP (a and c) or LFn-GFP (b and d) for 1 hr (a and b) or 2 hr (c and d) and imaged by confocal microscopy. (Right) Overlay of red and green staining. (Center) Uptake of transferrin in red. (Left) Internalized LFn-GFP or GFP in green. Similar results were found for COS cells (data not shown).

Article Snippet: HeLa cells (American Type Culture Collection), grown on collagen-treated chamber slides (BD Science) to reach ≈80% confluence, were incubated with 40 μg/ml purified GFP or LFn-GFP at 37°C for 1 or 2 h. Some incubations were performed in the presence of 100 μg/ml Texas red-conjugated transferrin (Molecular Probes) as a marker for the endocytic pathway or with 10 μg/ml PA. For the transferrin experiments, cells were washed four times with cold DMEM and then fixed for 15 min in 4% paraformaldehyde in cold PBS.

Techniques: Incubation, Confocal Microscopy, Staining

Journal:

Article Title: A fragment of anthrax lethal factor delivers proteins to the cytosol without requiring protective antigen

doi: 10.1073/pnas.1131930100

Figure Lengend Snippet: Internalized LFn-GFP does not completely localize with the endocytic or secretory pathways. HeLa cells incubated for 1 hr with LFn-GFP in the absence of PA were stained with markers for early endosomes (EEA-1; a), late endosomes (Lamp-1; b), lysosomes (Lamp-2; c), and the Golgi apparatus (Ab-1; d) and visualized by confocal microscopy. (Right) Overlay of red and green staining. (Center) Red organelle antibody staining. (Left) Green fluorescence of LFn-GFP.

Article Snippet: HeLa cells (American Type Culture Collection), grown on collagen-treated chamber slides (BD Science) to reach ≈80% confluence, were incubated with 40 μg/ml purified GFP or LFn-GFP at 37°C for 1 or 2 h. Some incubations were performed in the presence of 100 μg/ml Texas red-conjugated transferrin (Molecular Probes) as a marker for the endocytic pathway or with 10 μg/ml PA. For the transferrin experiments, cells were washed four times with cold DMEM and then fixed for 15 min in 4% paraformaldehyde in cold PBS.

Techniques: Incubation, Staining, Confocal Microscopy, Fluorescence

Journal:

Article Title: A fragment of anthrax lethal factor delivers proteins to the cytosol without requiring protective antigen

doi: 10.1073/pnas.1131930100

Figure Lengend Snippet: Colocalization of LFn-GFP with the proteosome 20s subunit. HeLa cells were stained with the mixture of an antibody to the α-subunit and the other to the β-subunit of the 20s proteosome 2 hr after incubation with LFn-GFP. Similar results were also found after 1 hr incubation (see Fig. 1) and with a single antibody to the α-subunit or β-subunit of the proteosome, respectively (data not shown). (Top) Green fluorescence of LFn-GFP. (Middle) Red fluorescence of the proteosome antibody. (Bottom) Overlay of both channels.

Article Snippet: HeLa cells (American Type Culture Collection), grown on collagen-treated chamber slides (BD Science) to reach ≈80% confluence, were incubated with 40 μg/ml purified GFP or LFn-GFP at 37°C for 1 or 2 h. Some incubations were performed in the presence of 100 μg/ml Texas red-conjugated transferrin (Molecular Probes) as a marker for the endocytic pathway or with 10 μg/ml PA. For the transferrin experiments, cells were washed four times with cold DMEM and then fixed for 15 min in 4% paraformaldehyde in cold PBS.

Techniques: Staining, Incubation, Fluorescence

Journal:

Article Title: A fragment of anthrax lethal factor delivers proteins to the cytosol without requiring protective antigen

doi: 10.1073/pnas.1131930100

Figure Lengend Snippet: Internalization and colocalization of LFn-GFP with the proteosome 20s subunit is more efficient in the absence of PA. HeLa cells were stained for the proteosome 20s α-subunit 1 hr (a and b)or2hr(c and d) after incubation with LFn-GFP in the absence (a and c) or presence (b and d) of PA. (Right) Overlay. (Center) Red fluorescent staining for the proteosome. (Left) Green fluorescence of LFn-GFP.

Article Snippet: HeLa cells (American Type Culture Collection), grown on collagen-treated chamber slides (BD Science) to reach ≈80% confluence, were incubated with 40 μg/ml purified GFP or LFn-GFP at 37°C for 1 or 2 h. Some incubations were performed in the presence of 100 μg/ml Texas red-conjugated transferrin (Molecular Probes) as a marker for the endocytic pathway or with 10 μg/ml PA. For the transferrin experiments, cells were washed four times with cold DMEM and then fixed for 15 min in 4% paraformaldehyde in cold PBS.

Techniques: Staining, Incubation, Fluorescence