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1.
FIGURE 1.

FIGURE 1. From: Niemann-Pick Type C1 I1061T Mutant Encodes a Functional Protein That Is Selected for Endoplasmic Reticulum-associated Degradation Due to Protein Misfolding.

Immunofluorescence staining for NPC1 (A and C) and filipin staining for cholesterol (B and D) in WT (A and B) and NPC1I1061T mutant fibroblasts (C and D). Bar, 50 μm.

Mark E. Gelsthorpe, et al. J Biol Chem. 2008 March 28;283(13):8229-8236.
2.
FIGURE 7.

FIGURE 7. From: Niemann-Pick Type C1 I1061T Mutant Encodes a Functional Protein That Is Selected for Endoplasmic Reticulum-associated Degradation Due to Protein Misfolding.

Time course of complementation of npc1-null CHO cells by transient expression of WT npc1-GFP or npc1I1061T-GFP. Graphs show the extent of complementation of npc1-null CHO cells by the WT and npc1I1061T constructs at 24 (A), 48 (B), and 72 (C) h post-transfection. Transfected cells (GFP-positive) were either scored as none (<20% complementation), partial (20–80% complementation), or complete (>80% complementation). p = NS for WT npc1-GFP versus npc1I1061T-GFP at 72 h. D, comparison of the extent of complementation of npc1-null CHO cells by the npc1I1061T and npc1I1061T-GFP constructs at 72 h post-transfection. p = NS for npc1I1061T versus npc1I1061T-GFP.

Mark E. Gelsthorpe, et al. J Biol Chem. 2008 March 28;283(13):8229-8236.
3.
FIGURE 4.

FIGURE 4. From: Niemann-Pick Type C1 I1061T Mutant Encodes a Functional Protein That Is Selected for Endoplasmic Reticulum-associated Degradation Due to Protein Misfolding.

Metabolic labeling of newly synthesized NPC1 protein followed by Endo H treatment. A, WT and NPC1I1061T mutant, and B, fibroblasts were pulse-labeled as above, followed by 0–8 h chase, IP with NPC1 antibody, and Endo H treatment. U, untreated, P, peptide:N-glycosidase F treated, and C, IP in the presence of excess immunizing peptide. Closed arrows and open arrows indicate Endo H-sensitive and Endo H-resistant species, respectively. Graphs show densitometric values for Endo H-sensitive (gray bars) and Endo H-resistant species (black bars) in WT (upper right panel) and NPC1I1061T mutant fibroblasts (lower right panel).

Mark E. Gelsthorpe, et al. J Biol Chem. 2008 March 28;283(13):8229-8236.
4.
FIGURE 5.

FIGURE 5. From: Niemann-Pick Type C1 I1061T Mutant Encodes a Functional Protein That Is Selected for Endoplasmic Reticulum-associated Degradation Due to Protein Misfolding.

NPC1I1061T protein is proteasomally degraded. A, NPC1I1061T cells were treated for 24 h in 10% glycerol or incubated at 26 °C, and cell lysates were subjected to Western blot analysis for NPC1 expression. A representative blot is shown and the graph displays NPC1 expression normalized toβ-actin. *, p < 0.05 for 26 °C versus untreated, p = 0.057 for glycerol versus untreated. B, NPC1I1061T cells were treated for 24 h with 20μm PBA, and lysates were subjected to Western blot analysis for NPC1 expression. A representative blot is shown and the graph displays NPC1 expression normalized to β-actin. *, p < 0.05 for PBA versus untreated. C, NPC1I1061T cells were treated for 24 h with the proteasome inhibitor MG132 (50 μm) or with the lysosome inhibitor chloroquine (10 μm), and lysates were subjected to Western blot analysis for NPC1 expression. A representative blot is shown and the graph displays NPC1 expression normalized to β-actin. *, p < 0.05 for MG132 versus untreated.

Mark E. Gelsthorpe, et al. J Biol Chem. 2008 March 28;283(13):8229-8236.
5.
FIGURE 2.

FIGURE 2. From: Niemann-Pick Type C1 I1061T Mutant Encodes a Functional Protein That Is Selected for Endoplasmic Reticulum-associated Degradation Due to Protein Misfolding.

Characterization of NPC1 expression in human fibroblasts homozygous for NPC1I1061T mutations. Data shown are for three independent WT and three independent NPC1I1061T human fibroblast cell lines. A, real-time quantitative PCR for NPC1 mRNA levels in WT (black bars) and NPC1I1061T mutant fibroblasts (gray bars). NPC1 mRNA expression is normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA. *, p = 0.056 for mean WT versus mean NPC1I1061T. B, Western blot analysis for NPC1 protein expression in WT and NPC1I1061T mutant fibroblasts. Note 4-fold greater loading of protein in NPC1I1061T lanes compared with WT lanes. NPC1 expression is normalized to β-actin expression (5 μg/lane). C, quantification of NPC1 protein expression in WT (black bars) and NPC1I1061T mutant fibroblasts (gray bars). *, p < 0.01 for mean WT versus mean NPC1I1061T.

Mark E. Gelsthorpe, et al. J Biol Chem. 2008 March 28;283(13):8229-8236.
6.
FIGURE 8.

FIGURE 8. From: Niemann-Pick Type C1 I1061T Mutant Encodes a Functional Protein That Is Selected for Endoplasmic Reticulum-associated Degradation Due to Protein Misfolding.

Overexpression of NPC1I1061T rescues the cholesterol esterification defect in npc1-null cells. A, Western blot analysis of NPC1 and p63 protein expression in microsomes (5 μg/lane) isolated from WT CHO cells; npc1-null CHO cells; npc1-null cell lines expressing WT NPC1, NPC1I1061T, and NPC1P692S; and WT mouse embryonic fibroblasts. The lower Mr for NPC1 protein in WT mouse embryonic fibroblasts likely represents differences in glycosylation patterns between the hamster and murine cell lines. B, quantification of NPC1 protein in npc1-null cell lines expressing WT NPC1, NPC1I1061T, and NPC1P692S. NPC1 expression is normalized to expression of p63, a resident ER membrane protein, and densitometry presented as mean of duplicate lanes. C, LDL-stimulated cholesterol esterification in WT CHO cells; npc1-null CHO cells; and npc1-null cell lines expressing WT NPC1, NPC1I1061T, and NPC1P692S. Assays were performed in triplicate and values represent mean ± S.E. *, p < 0.05 for mean cholesterol esterification in npc1-null cells versus npc1I1061T cells.

Mark E. Gelsthorpe, et al. J Biol Chem. 2008 March 28;283(13):8229-8236.
7.
FIGURE 6.

FIGURE 6. From: Niemann-Pick Type C1 I1061T Mutant Encodes a Functional Protein That Is Selected for Endoplasmic Reticulum-associated Degradation Due to Protein Misfolding.

Overexpression of NPC1I1061T rescues the cholesterol accumulation phenotype in npc1-null cells. A, npc1-deficient CHO cells were transiently transfected with either npc1I1061T-GFP (top panels) or npc1P692S-GFP (bottom panels) constructs and stained with filipin for unesterified cholesterol. Cells were examined by immunofluorescence for cholesterol (left panels) and GFP expression (right panels). npc1-null cells expressing npc1I1061T-GFP (top panels, closed arrows) are filipin-negative, whereas non-transfected cells (top panels, open arrows) are filipin-positive indicative of NPC1 mutant phenotype. npc1-null cells expressing the non-functional mutant npc1P692Sbottom panels, closed arrows) remain filipin-positive. Bar, 10 μm. B, NPC1-deficient human fibroblasts were transiently transfected with either GFP (left panel) or npc1I1061T-GFP (right panel) constructs and tested for the ability to complement the NPC1 phenotype. Merged immunofluorescence images are shown for GFP (green) and cholesterol (blue) staining. In the right panel, npc1-null cells expressing npc1I1061T-GFP (closed arrows) are complemented (low cholesterol staining), whereas in the left panel GFP expressing cells (closed arrows) are not complemented (high cholesterol staining). Non-transfected cells (open arrows) do not exhibit complementation. Bar, 50 μm.

Mark E. Gelsthorpe, et al. J Biol Chem. 2008 March 28;283(13):8229-8236.
8.
FIGURE 3.

FIGURE 3. From: Niemann-Pick Type C1 I1061T Mutant Encodes a Functional Protein That Is Selected for Endoplasmic Reticulum-associated Degradation Due to Protein Misfolding.

Regulation of NPC1I1061T protein expression by ER quality control. A and B, metabolic labeling of newly synthesized NPC1 protein. WT (A) and NPC1I1061T mutant (B) fibroblasts were pulsed for 1 h with [35S]Cys/Met containing media followed by 0–72 h (WT) or 0–12 h (NPC1I1061T) chase and IP with NPC1 antibody. For each time point two independent samples are shown. Left lanes (0+comp) show IP in the presence of excess immunizing peptide (competition). Closed arrows identify nonspecific band. Note the shift to higher molecular weight species (open arrows) in WT cells from 0 to 4 h, which is absent in the NPC1I1061T mutant. C, the graph plots densitometric values for radiolabeled NPC1 versus time. From 0 to 8 h both WT (blue line) and NPC1I1061T fibroblasts (red line) show similar initial rapid rates of NPC1 protein degradation (WT t½ = 9 h, NPC1I1061T t½ = 6.5 h), whereas from 8 to 72 h WT (blue line) NPC1 protein degradation is decreased to t½ = 42 h.

Mark E. Gelsthorpe, et al. J Biol Chem. 2008 March 28;283(13):8229-8236.

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