rabbit polyclonal anti npc1  (Novus Biologicals)

Journal: Chemistry & biology

Article Title: Discovery of oxysterol-derived pharmacological chaperones for NPC1: implication for the existence of second sterol-binding site.

doi: 10.1016/j.chembiol.2013.02.009

Figure Lengend Snippet: Figure 2. Identification of Sterol Derivatives with Greater Potency by Means of Chemical Optimization (A) Structures of representative sterol derivatives used in this article. (B) NPC1I1061T colocalization assay, showing dose-response curves for selected oxysterols and sterol derivatives. The extent of colocalization of the NPC1 mutant and LAMP1 was quantified as described in Experimental Procedures, and the data points represent the averages (n = 10) with SE depicted by error bars. (C) Representative images of the experiments in (B). Calibration bar represents 20 mm. See also Figure S1A.

Article Snippet: For immunoblotting of endogenous NPC1 protein, rabbit anti-NPC1 polyclonal antibody (Novus Biologicals) combined with HRP-conjugated anti-rabbit antibody (R&D Systems) was used.

Techniques: Mutagenesis

Journal: Chemistry & biology

Article Title: Discovery of oxysterol-derived pharmacological chaperones for NPC1: implication for the existence of second sterol-binding site.

doi: 10.1016/j.chembiol.2013.02.009

Figure Lengend Snippet: Figure 3. Effect of Oxysterol Derivatives on Steady-State Expression Level and Maturation Status of NPC1I1061T Mutant (A) The steady-state expression level of FLAG-NPC1-GFP (WT or I1061T) was quantified by measuring GFP fluorescence in the lysate with or without oxysterol derivatives. The GFP fluorescence was normalized with respect to total protein concentration. Data points represent the averages (n = 3) with SD depicted by error bars. (B) Acquisition of EndoH resistance upon treatment with 25HC and its derivative. Cells stably expressing either WT or I1061T version of FLAG-NPC1-GFP were treated as indicated for 24 hr and lysed. The lysates were digested with EndoH and immunoprecipitated with anti-FLAG beads. The immunoprecipitated proteins were subjected to western blot analysis (immunoblotted with anti-FLAG antibody).

Article Snippet: For immunoblotting of endogenous NPC1 protein, rabbit anti-NPC1 polyclonal antibody (Novus Biologicals) combined with HRP-conjugated anti-rabbit antibody (R&D Systems) was used.

Techniques: Expressing, Mutagenesis, Protein Concentration, Stable Transfection, Immunoprecipitation, Western Blot

Journal: Chemistry & biology

Article Title: Discovery of oxysterol-derived pharmacological chaperones for NPC1: implication for the existence of second sterol-binding site.

doi: 10.1016/j.chembiol.2013.02.009

Figure Lengend Snippet: Figure 5. Functional Rescue of Patient- Derived Fibroblasts (A) Comparison of the expression levels and band patterns of endogenous WT (HEK293) and I1061T NPC1 proteins (NPC fibroblast). The filled arrow- head indicates the mature form, and the open arrowhead indicates the immature form. (B) Effects of 25HC and mo56HC on expression level and band pattern of endogenous NPC1I1061T. NPC fibroblasts were treated with the indicated compound for 48 hr and processed for western blot analysis using anti-NPC1 antibody. (C) Effect of other sterol derivatives on expression level and band pattern of I1061T mutant. To facil- itate comparison between the compounds, the concentrations normalized with their EC50s are also shown. (D) Alleviation of intracellular cholesterol accumu- lation by oxysterol derivative. NPC fibroblasts were cultured in the presence of the indicated compound for 48 hr, and processed for filipin staining. Calibration bar represents 100 mm. The intracellular cholesterol accumulation was quanti- fied as described in Experimental Procedures. Error bar represents SD (n = 12). See also Figure S2.

Article Snippet: For immunoblotting of endogenous NPC1 protein, rabbit anti-NPC1 polyclonal antibody (Novus Biologicals) combined with HRP-conjugated anti-rabbit antibody (R&D Systems) was used.

Techniques: Functional Assay, Derivative Assay, Comparison, Expressing, Western Blot, Mutagenesis, Cell Culture, Staining

Journal: Chemistry & biology

Article Title: Discovery of oxysterol-derived pharmacological chaperones for NPC1: implication for the existence of second sterol-binding site.

doi: 10.1016/j.chembiol.2013.02.009

Figure Lengend Snippet: Figure 6. Dispensability of NTD for Oxysterol Derivative-Mediated Rescue of Mutant NPC1 Protein (A) Schematic representation of the NTD-deleted NPC1-GFP (DNTD). (B) Subcellular localization of the DNTD-WT and DNTD-I1061T. Cells stably expressing the DNTD-NPC1-GFP construct were treated as indicated for 24 hr and colocalization of the NPC1 with LAMP1 was examined. Calibration bar represents 20 mm. (C) Dose-dependent rescue of DNTD-I1061T localization by representative oxysterol derivatives. The graph shows the dose-response curves of representative compounds and the table shows calculated EC50 values. For 25HC, the extrapolated value is shown. Error bar, SE (n = 12).

Article Snippet: For immunoblotting of endogenous NPC1 protein, rabbit anti-NPC1 polyclonal antibody (Novus Biologicals) combined with HRP-conjugated anti-rabbit antibody (R&D Systems) was used.

Techniques: Mutagenesis, Stable Transfection, Expressing, Construct

Journal: Chemistry & biology

Article Title: Discovery of oxysterol-derived pharmacological chaperones for NPC1: implication for the existence of second sterol-binding site.

doi: 10.1016/j.chembiol.2013.02.009

Figure Lengend Snippet: Figure 7. Existence of Non-NTD Sterol-Binding Site (A) Sterol-mediated stabilization of DNTD-I1061T. The steady-state expression level of DNTD-I1061T was quantified as in Figure 3A. Error bar, SD (n = 3). (B) Schematic representation of the NTD-tail-GFP construct. See also Figure S3. (C) Photoaffinity labeling experiments of NTD-deleted NPC1 and NTD-tail NPC1. Membranes from cells stably expressing either FLAG-tagged DNTD-I1061T or NTD-tail-GFP were labeled with mo56AZK as in Figure 4. Right panel shows the labeling of DNTD-WT. Because of the low expression level of the stable cell line, the longer exposure time was used for DNTD-WT. ns, nonspecific labeling/staining. See also Figure S3C. (D) The quantified results of (C).

Article Snippet: For immunoblotting of endogenous NPC1 protein, rabbit anti-NPC1 polyclonal antibody (Novus Biologicals) combined with HRP-conjugated anti-rabbit antibody (R&D Systems) was used.

Techniques: Binding Assay, Expressing, Construct, Labeling, Stable Transfection, Staining