texas red labeled transferrin  (Thermo Fisher)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Mechanistic analysis of a novel membrane-interacting variable loop in the pleckstrin-homology domain critical for dynamin function.

doi: 10.1073/pnas.2215250120

Figure Lengend Snippet: Fig. 4. Overexpression of VL4 mutants cause defects in clathrin-mediated endocytosis. (A) Representative micrographs showing uptake of fluorescent transferrin in dynamin2 KO HeLa cells expressing Dyn1-GFP and its mutants. (B) Tx-Red transferrin uptake in cells expressing Dyn1-GFP (n = 84), Dyn1(K44A)-GFP (n = 57), Dyn1(F579A)-GFP (n = 109), and Dyn1(M580T)-GFP (n = 58). Data were normalized to the mean transferrin fluorescence seen in non-transfected cells for each condition. Significance was estimated using Mann–Whitney’s test where **** denotes P < 0.0001.

Article Snippet: The media was exchanged for HEPES-buffered Hank’s balanced salt solution, and cells were fed with 50 μg mL−1 of Texas Red-labeled transferrin (Invitrogen) and incubated for 10 min.

Techniques: Over Expression, Expressing, Fluorescence, Transfection

Journal: Nature Communications

Article Title: Rapid cell-surface prion protein conversion revealed using a novel cell system

doi: 10.1038/ncomms1282

Figure Lengend Snippet: ( a ) PrP224-AlaMYC cells were exposed to RML prions for the indicated durations, and then fixed and extracted with formic acid before staining with anti-MYC antibodies (green) and counterstaining with 6-diamidino-2-phenylindole (DAPI, blue). Orthogonal projections (red and green squares) of serial confocal sections are shown alongside one z-section taken from the middle of the cell (as indicated by the guidelines). A cell fixed after a 1-min exposure to RML prions shows PrP Sc immunostaining only at the cell surface (1 min, white arrow). An adjacent cell in the field that has high levels of plasma membrane PrP Sc also shows low intracellular levels of PrP Sc (1 min, yellow arrow). Cells exposed to RML prions for 180 min show the typical steady-state distribution of PrP Sc , with strong immunostaining at the plasma membrane (white arrows) and in the perinuclear region (yellow arrow −180 min); scale bar, 10 μm. ( b ) Quantification of cell phenotypes observed at different time points following prion exposure (the mean±s.e.m. from four independent experiments are shown; * P <0.05, ** P <0.01, two-tailed t -test. The exact P -values are: membrane 1–2 min, 0.022; membrane 1–4 min, 0.049; membrane/PNC 1–2 min, 0.0049; membrane PNC 1–4 min, 0.0057). ( c ) PrP-224AlaMYC cells were exposed to RML prions and Texas red-labelled transferrin (red) for the indicated durations, then fixed and extracted with formic acid. Merged confocal images of cells stained with anti-MYC antibodies (green) and counterstained with DAPI (blue) are shown; scale bar, 20 μm. Initially, PrP Sc shows a plasma membrane distribution (1 min, arrows) and/or diffuse intracellular distribution (2 min, arrows) and then rapidly attains its steady-state distribution concentrated at the plasma membrane and PNC (4 min, white arrow indicates strong PNC stain). Initially, transferrin could be observed in small puncta at the cell periphery, reaching its equilibrium distribution in recycling endosomes at the PNC (yellow arrows) between 8 and 16 min.

Article Snippet: Texas red-labelled transferrin (Invitrogen) was added to the media at 5 μg ml −1 for the last 10 min of the incubation.

Techniques: Staining, Immunostaining, Two Tailed Test

Journal: Nature Communications

Article Title: Rapid cell-surface prion protein conversion revealed using a novel cell system

doi: 10.1038/ncomms1282

Figure Lengend Snippet: ( a ) Pre-cooled PrP-224AlaMYC cells were exposed to RML prions for 2 min on ice, then fixed and extracted with formic acid. Orthogonal reconstructions of serial confocal slices are shown (red and green boxes) alongside one z-slice taken from near the middle of the cells (as indicated by the guide lines). The cells were fixed after 2 min and stained with anti-MYC antibodies (green) and counterstained with 6-diamidino-2-phenylindole (DAPI, blue); scale bar, 20 μm. Formic acid-resistant PrP (PrP Sc ) is formed on the cell surface of one of the cells in the field (arrow), no intracellular PrP Sc was detected. ( b ) PrP-224AlaMYC cells were mock-transfected or transfected with RNAi directed at CHC, pretreated with dynasore (80 μM), EIPA (100 μM) or pre-cooled to 4 °C. RML prions were added in the continued presence of the treatments for 180 min and Texas red-labelled transferrin (red) was added for the final 10 min of incubation. The cells were then fixed and extracted with formic acid. Merged confocal images of cells stained with anti-MYC antibodies (green) and counterstained with DAPI (blue) are shown; scale bar, 20 μm. Following prion exposure at 4 °C, MYC-tagged PrP Sc was detected only at the plasma membrane (arrow). ( c ) Lysates from PrP-224AlaMYC cells mock-transfected or transfected with RNAi directed at CHC were collected and subjected to SDS–polyacrylamide gel electrophoresis. Immunoblots developed with anti-CHC and anti-actin antibodies are shown. Quantitative densitometry of similar blots showed that CHC expression was reduced by ∼70% in the RNAi-treated cells. ( d ) PrP-224AlaMYC cells were treated as in b . The percentage of anti-MYC-positive (RML prion infected) cells observed was quantified for each condition. The mean±s.e.m. from three independent experiments are shown (background levels found in uninfected cells for each condition have been subtracted from the mean).

Article Snippet: Texas red-labelled transferrin (Invitrogen) was added to the media at 5 μg ml −1 for the last 10 min of the incubation.

Techniques: Staining, Transfection, Incubation, Polyacrylamide Gel Electrophoresis, Western Blot, Expressing, Infection