Journal: Developmental cell

Article Title: NPC1-mTORC1 signaling Couples Cholesterol Sensing to Organelle Homeostasis and is a Targetable Pathway in Niemann-Pick type C

doi: 10.1016/j.devcel.2020.11.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal anti-CLN2 (TPP1) , Santa Cruz Biotechnology , Cat#sc-393961.

Techniques: Labeling, Plasmid Preparation, Recombinant, Control, Software, Magnetic Beads

Journal: Science Advances

Article Title: Exploiting the diphtheria toxin internalization receptor enhances delivery of proteins to lysosomes for enzyme replacement therapy

doi: 10.1126/sciadv.abb0385

Figure Lengend Snippet: ( A ) Design of LTM-TPP1 fusion protein and delivery schematic. ( B ) Enzyme kinetics of rTPP1 and LTM-TPP1 against the synthetic substrate AAF-AMC are indistinguishable. Michaelis-Menten plots were generated by varying [AAF-AMC] at a constant concentration of 10 nM enzyme (means ± SD; n = 3). Plots and kinetic parameters were calculated with GraphPad Prism 7.04. ( C ) Maturation of TPP1 is unaffected by the N-terminal fusion of LTM. ( D ) LTM-TPP1 inhibits wild-type DT activity in a dose-dependent manner (IC 50 of 17.2 nM), while rTPP1 has no effect on protein synthesis inhibition by DT (means ± SD; n = 3). ( E ) LTM and DTR-TPP1 bind HBEGF with apparent K d ’s of 13.3 and 19.1 nM, respectively. ( F ) LTM-TPP1 colocalizes with LAMP1 staining (red).

Article Snippet: Membranes were then blocked for 1 hour with a 5% milk–tris-buffered saline (TBS) solution and incubated overnight at room temperature with a 1:100 dilution of mouse monoclonal antibody against TPP1 (Abcam, ab54685) in 5% milk-TBS.

Techniques: Generated, Concentration Assay, Activity Assay, Inhibition, Staining

Journal: Science Advances

Article Title: Exploiting the diphtheria toxin internalization receptor enhances delivery of proteins to lysosomes for enzyme replacement therapy

doi: 10.1126/sciadv.abb0385

Figure Lengend Snippet: ( A ) CLN2 knockout cells exhibit ~4% TPP1 activity relative to wild-type HeLa Kyoto cells (means ± SD; n = 3). ( B ) Western blotting against TPP1 reveals no detectable protein in the knockout cells. ( C ) (Left) In vitro maturation of pro-rTPP1 and LTM-TPP1 (16 ng) was analyzed by Western blot. (Right) TPP1 present in wild-type (WT) and TPP1 −/− cells, and TPP1 −/− cells treated with 100 nM rTPP1 and LTM-TPP1. ( D ) Uptake of rTPP1 and LTM-TPP1 into HeLa Kyoto TPP1 −/− cells was monitored by TPP1 activity (means ± SD; n = 4). ( E ) TPP1 activity present in HeLa Kyoto TPP1 −/− cells following a single treatment with 50 nM LTM-TPP1 (means ± SD; n = 3).

Article Snippet: Membranes were then blocked for 1 hour with a 5% milk–tris-buffered saline (TBS) solution and incubated overnight at room temperature with a 1:100 dilution of mouse monoclonal antibody against TPP1 (Abcam, ab54685) in 5% milk-TBS.

Techniques: Knock-Out, Activity Assay, Western Blot, In Vitro

Journal: Science Advances

Article Title: Exploiting the diphtheria toxin internalization receptor enhances delivery of proteins to lysosomes for enzyme replacement therapy

doi: 10.1126/sciadv.abb0385

Figure Lengend Snippet: Uptake of rTPP1 and LTM-TPP1 into HeLa Kyoto TPP1 −/− cells following EndoH treatment to remove N -glycan labeling (means ± SD; n = 3).

Article Snippet: Membranes were then blocked for 1 hour with a 5% milk–tris-buffered saline (TBS) solution and incubated overnight at room temperature with a 1:100 dilution of mouse monoclonal antibody against TPP1 (Abcam, ab54685) in 5% milk-TBS.

Techniques: Glycoproteomics, Labeling

Journal: Science Advances

Article Title: Exploiting the diphtheria toxin internalization receptor enhances delivery of proteins to lysosomes for enzyme replacement therapy

doi: 10.1126/sciadv.abb0385

Figure Lengend Snippet: ( A ) Assay schematic. ( B ) TPP1 activity in brain homogenates of 6-week-old mice injected with two doses (5 and 25 μg) of either rTPP1 or LTM-TPP1 (5 μg, P = 0.01; 25 μg, P = 0.002). ( C ) TPP1 activity in brain homogenates following a single 25-μg dose of LTM-TPP1, 1, 7, and 14 days postinjection. Data are presented as box and whisker plots, with whiskers representing minimum and maximum values from n ≥ 4 mice per group. Statistical significance was calculated using paired t tests with GraphPad Prism 7.04.

Article Snippet: Membranes were then blocked for 1 hour with a 5% milk–tris-buffered saline (TBS) solution and incubated overnight at room temperature with a 1:100 dilution of mouse monoclonal antibody against TPP1 (Abcam, ab54685) in 5% milk-TBS.

Techniques: Activity Assay, Injection, Whisker Assay