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Novus Biologicals rabbit polyclonal anti npc1
Rabbit Polyclonal Anti Npc1, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 94/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 94 stars, based on 69 article reviews

rabbit polyclonal anti npc1 - by Bioz Stars, 2026-07

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Genotype and clinical and biochemical phenotype of Niemann-Pick C patients analyzed in this study.

Journal: Journal of Clinical Medicine

Article Title: Molecular Genetics of Niemann–Pick Type C Disease in Italy: An Update on 105 Patients and Description of 18 NPC1 Novel Variants

doi: 10.3390/jcm9030679

Figure Lengend Snippet: Genotype and clinical and biochemical phenotype of Niemann-Pick C patients analyzed in this study.

Article Snippet: After overnight blocking with 5% nonfat dry milk in PBS-Tween 0.1% (PBS-T), the membranes were probed with anti NPC1 polyclonal antibody (Novus Biologicals, Littleton, USA; NB400-148) overnight at 4 °C.

Techniques: Variant Assay

Relative distribution of the clinical phenotypes of Niemann–Pick type C1 (NPC1) patients based on the age at onset of first neurological signs. Severe infantile (SI): age at onset <2 years; Late infantile (LI): age at onset 2–6 years; Juvenile (J): age at onset 6–15 years; and Adult (A): age at onset ≥15 years. Patients who died during the first month of life due to liver or respiratory insufficiency without signs of neurological involvement were classified as early infantile systemic lethal form (EISL). NC: not classifiable.

Journal: Journal of Clinical Medicine

Article Title: Molecular Genetics of Niemann–Pick Type C Disease in Italy: An Update on 105 Patients and Description of 18 NPC1 Novel Variants

doi: 10.3390/jcm9030679

Figure Lengend Snippet: Relative distribution of the clinical phenotypes of Niemann–Pick type C1 (NPC1) patients based on the age at onset of first neurological signs. Severe infantile (SI): age at onset <2 years; Late infantile (LI): age at onset 2–6 years; Juvenile (J): age at onset 6–15 years; and Adult (A): age at onset ≥15 years. Patients who died during the first month of life due to liver or respiratory insufficiency without signs of neurological involvement were classified as early infantile systemic lethal form (EISL). NC: not classifiable.

Techniques:

Spectrum of NPC1 type of mutation mutations.

Journal: Journal of Clinical Medicine

Article Title: Molecular Genetics of Niemann–Pick Type C Disease in Italy: An Update on 105 Patients and Description of 18 NPC1 Novel Variants

doi: 10.3390/jcm9030679

Figure Lengend Snippet: Spectrum of NPC1 type of mutation mutations.

Techniques: Mutagenesis

Frequency of NPC1 mutant alleles identified in more than one allele.

Journal: Journal of Clinical Medicine

Article Title: Molecular Genetics of Niemann–Pick Type C Disease in Italy: An Update on 105 Patients and Description of 18 NPC1 Novel Variants

doi: 10.3390/jcm9030679

Figure Lengend Snippet: Frequency of NPC1 mutant alleles identified in more than one allele.

Techniques: Mutagenesis, Variant Assay

Clinical, biochemical, and molecular characteristics of the identified NPC1 novel mutations.

Journal: Journal of Clinical Medicine

Article Title: Molecular Genetics of Niemann–Pick Type C Disease in Italy: An Update on 105 Patients and Description of 18 NPC1 Novel Variants

doi: 10.3390/jcm9030679

Figure Lengend Snippet: Clinical, biochemical, and molecular characteristics of the identified NPC1 novel mutations.

Techniques: Staining, Variant Assay

NPC1 protein abundance in patients carrying seven novel missense mutations. ( A ) Representative western blot analysis of NPC1 protein expression in NPC patients and normal control fibroblast cell lines. ( B ) The intensity of the NPC1 signals was normalized against actin. The NPC1 protein content in NPC fibroblasts was expressed as a percentage of the NPC1 protein content found in fibroblasts from a normal control.

Journal: Journal of Clinical Medicine

Article Title: Molecular Genetics of Niemann–Pick Type C Disease in Italy: An Update on 105 Patients and Description of 18 NPC1 Novel Variants

doi: 10.3390/jcm9030679

Figure Lengend Snippet: NPC1 protein abundance in patients carrying seven novel missense mutations. ( A ) Representative western blot analysis of NPC1 protein expression in NPC patients and normal control fibroblast cell lines. ( B ) The intensity of the NPC1 signals was normalized against actin. The NPC1 protein content in NPC fibroblasts was expressed as a percentage of the NPC1 protein content found in fibroblasts from a normal control.

Techniques: Quantitative Proteomics, Western Blot, Expressing, Control

Functional analysis of four NPC1 splicing mutations. ( A ) The c.181-2A>G mutation caused the creation of a novel acceptor splice site leading to the insertion of a nucleotide in the mRNA transcript and the alteration of the reading frame; ( B ) the c.3591+121C>T and ( C ) the c.3591+105A>T mutations led to the generation of a cryptic acceptor splice site within intron 23, causing the retention of 119 and 103 nt, respectively, the shifting in the open reading frame, and the generation of a premature stop codon. The abnormal transcript resulting from the presence of c.3591+121C>T mutation was degraded via non-sense mediated decay (NMD). ( D ) In patient NP67, the skipping of exon 14 was observed (r.2131_2245del). The abnormal transcript was degraded via NMD.

Journal: Journal of Clinical Medicine

Article Title: Molecular Genetics of Niemann–Pick Type C Disease in Italy: An Update on 105 Patients and Description of 18 NPC1 Novel Variants

doi: 10.3390/jcm9030679

Figure Lengend Snippet: Functional analysis of four NPC1 splicing mutations. ( A ) The c.181-2A>G mutation caused the creation of a novel acceptor splice site leading to the insertion of a nucleotide in the mRNA transcript and the alteration of the reading frame; ( B ) the c.3591+121C>T and ( C ) the c.3591+105A>T mutations led to the generation of a cryptic acceptor splice site within intron 23, causing the retention of 119 and 103 nt, respectively, the shifting in the open reading frame, and the generation of a premature stop codon. The abnormal transcript resulting from the presence of c.3591+121C>T mutation was degraded via non-sense mediated decay (NMD). ( D ) In patient NP67, the skipping of exon 14 was observed (r.2131_2245del). The abnormal transcript was degraded via NMD.

Techniques: Functional Assay, Mutagenesis

NPC1 protein structure and variants. The structure of NPC1 (PDB 5U73) showed the location of the NPC1 variants identified in this study. Functional domains containing NPC1 variants are indicated in different colors. MLD, middle luminal domain = green; CTD, C-terminal domain = yellow; TM5 = blue; Cytosolic loop = red; lumenal loop between TM5 and TM6 = pink.

Journal: Journal of Clinical Medicine

Article Title: Molecular Genetics of Niemann–Pick Type C Disease in Italy: An Update on 105 Patients and Description of 18 NPC1 Novel Variants

doi: 10.3390/jcm9030679

Figure Lengend Snippet: NPC1 protein structure and variants. The structure of NPC1 (PDB 5U73) showed the location of the NPC1 variants identified in this study. Functional domains containing NPC1 variants are indicated in different colors. MLD, middle luminal domain = green; CTD, C-terminal domain = yellow; TM5 = blue; Cytosolic loop = red; lumenal loop between TM5 and TM6 = pink.

Techniques: Functional Assay

RIDα rescues cholesterol storage phenotype in NPC1-deficient cells but not in NPC2-mutant fibroblasts. (A) Confocal images of NPC1-mutant fibroblasts mock transfected or transfected with FLAG-RIDα and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (B) Confocal images of CT43 and CT43-RIDα cells stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (C) Confocal images of shNPC1 and shNPC1-RIDα cells stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (D) Confocal images of NPC2-mutant fibroblasts mock transfected or transfected with FLAG-RIDα and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (E) Confocal images of NPC1- or NPC2-mutant fibroblasts transfected with FLAG-RIDα and stained with antibodies to LBPA and FLAG-RIDα. Boxed areas, regions of the image that were magnified. Bars, 10 μm. Nu, nucleus.

Journal: Molecular Biology of the Cell

Article Title: Adenovirus RIDα uncovers a novel pathway requiring ORP1L for lipid droplet formation independent of NPC1

doi: 10.1091/mbc.E12-10-0760

Figure Lengend Snippet: RIDα rescues cholesterol storage phenotype in NPC1-deficient cells but not in NPC2-mutant fibroblasts. (A) Confocal images of NPC1-mutant fibroblasts mock transfected or transfected with FLAG-RIDα and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (B) Confocal images of CT43 and CT43-RIDα cells stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (C) Confocal images of shNPC1 and shNPC1-RIDα cells stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (D) Confocal images of NPC2-mutant fibroblasts mock transfected or transfected with FLAG-RIDα and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (E) Confocal images of NPC1- or NPC2-mutant fibroblasts transfected with FLAG-RIDα and stained with antibodies to LBPA and FLAG-RIDα. Boxed areas, regions of the image that were magnified. Bars, 10 μm. Nu, nucleus.

Article Snippet: The following antibodies were used: actin mouse monoclonal antibody (Sigma, St. Louis, MO); calreticulin chicken polyclonal antibody (Abcam, Cambridge, MA); E1A mouse monoclonal antibody (BD Biosciences, San Jose, CA); FLAG-M2 mouse monoclonal antibody and FLAG rat monoclonal antibody (Sigma); GFP rabbit polyclonal antibody (Abcam); hamster- and human-specific LAMP1 mouse monoclonal antibodies (Developmental Studies Hybridoma Bank, Iowa City, IA); LBPA mouse monoclonal antibody (Echelon Biosciences, Salt Lake City, UT); NPC1 rabbit polyclonal antibody (Novus Biologicals, Littleton, CO); ORP1L rabbit monoclonal antibody (Abcam); and RIDα rabbit polyclonal antibody produced as described in Hoffman et al. (1992 ).

Techniques: Mutagenesis, Transfection, Staining

NPC1 genotypes in three NPC1-deficient model systems used in this study.

Journal: Molecular Biology of the Cell

Article Title: Adenovirus RIDα uncovers a novel pathway requiring ORP1L for lipid droplet formation independent of NPC1

doi: 10.1091/mbc.E12-10-0760

Figure Lengend Snippet: NPC1 genotypes in three NPC1-deficient model systems used in this study.

Techniques: Expressing, Mutagenesis, shRNA, Knockdown

RIDα increases LD formation in NPC1-deficient cells. (A) Confocal images of normal and NPC1- and NPC2-mutant fibroblasts transfected with RIDα and stained with antibody to FLAG-RIDα and with BODIPY 493/503 and DAPI to visualize LDs and nuclei, respectively. Mock-transfected cells lacking FLAG-RIDα expression are shown in the same field and designated with an asterisk. (B) Confocal images of shControl, shNPC1, and shNPC1-RIDα cells stained with antibody to FLAG-RIDα and with BODIPY 493/503 and DAPI. (C) Confocal images of CT43 and CT43-RIDα cells stained with antibody to FLAG-RIDα and with BODIPY 493/503 and DAPI. (D, E) Quantification of average LD area (D) and average LD number (E) per cell in cells treated similarly to cells in C as described in Materials and Methods . Data are presented as mean ± SEM (* p < 0.001). (F) Quantification of esterified cholesterol in Chinese hamster ovary, CT43, and CT43-RIDα cells using the Amplex Red Cholesterol Assay kit as described in Materials and Methods . Values were normalized to total cellular protein and are displayed as mean ± SEM (* p < 0.001). (G) ACAT mRNA levels quantified by real-time PCR similarly to cells in . Data are presented as mean ± SEM. (H) Experimental setup of cholesterol transport assay. Purified human LDL was labeled with [ 3 H]cholesteryl palmitate, and cells were incubated with the labeled LDL and excess oleate. The labeled LDL was transported to Ly (step 1) and deesterified by lysosomal acid lipase (LAL; step 2). The liberated [ 3 H]cholesterol can then be transported to the ER (step 3), where it can be reesterified by ACAT along with the excess oleate to form [ 3 H]cholesteryl oleate and stored in LDs (step 4). (I) shControl, shNPC1, and shNPC1-RIDα cells were incubated with [ 3 H]cholesteryl palmitate along with excess oleate as described in Materials and Methods . The [ 3 H]cholesteryl oleate production was quantified, and values were normalized to total cellular protein and are displayed as mean ± SD (* p < 0.0001) from three independent experiments. Bars, 10 μm.

Journal: Molecular Biology of the Cell

Article Title: Adenovirus RIDα uncovers a novel pathway requiring ORP1L for lipid droplet formation independent of NPC1

doi: 10.1091/mbc.E12-10-0760

Figure Lengend Snippet: RIDα increases LD formation in NPC1-deficient cells. (A) Confocal images of normal and NPC1- and NPC2-mutant fibroblasts transfected with RIDα and stained with antibody to FLAG-RIDα and with BODIPY 493/503 and DAPI to visualize LDs and nuclei, respectively. Mock-transfected cells lacking FLAG-RIDα expression are shown in the same field and designated with an asterisk. (B) Confocal images of shControl, shNPC1, and shNPC1-RIDα cells stained with antibody to FLAG-RIDα and with BODIPY 493/503 and DAPI. (C) Confocal images of CT43 and CT43-RIDα cells stained with antibody to FLAG-RIDα and with BODIPY 493/503 and DAPI. (D, E) Quantification of average LD area (D) and average LD number (E) per cell in cells treated similarly to cells in C as described in Materials and Methods . Data are presented as mean ± SEM (* p < 0.001). (F) Quantification of esterified cholesterol in Chinese hamster ovary, CT43, and CT43-RIDα cells using the Amplex Red Cholesterol Assay kit as described in Materials and Methods . Values were normalized to total cellular protein and are displayed as mean ± SEM (* p < 0.001). (G) ACAT mRNA levels quantified by real-time PCR similarly to cells in . Data are presented as mean ± SEM. (H) Experimental setup of cholesterol transport assay. Purified human LDL was labeled with [ 3 H]cholesteryl palmitate, and cells were incubated with the labeled LDL and excess oleate. The labeled LDL was transported to Ly (step 1) and deesterified by lysosomal acid lipase (LAL; step 2). The liberated [ 3 H]cholesterol can then be transported to the ER (step 3), where it can be reesterified by ACAT along with the excess oleate to form [ 3 H]cholesteryl oleate and stored in LDs (step 4). (I) shControl, shNPC1, and shNPC1-RIDα cells were incubated with [ 3 H]cholesteryl palmitate along with excess oleate as described in Materials and Methods . The [ 3 H]cholesteryl oleate production was quantified, and values were normalized to total cellular protein and are displayed as mean ± SD (* p < 0.0001) from three independent experiments. Bars, 10 μm.

Techniques: Mutagenesis, Transfection, Staining, Expressing, Amplex Red Cholesterol Assay, Real-time Polymerase Chain Reaction, Transport Assay, Purification, Labeling, Incubation

Inhibition of ACAT blocks RIDα rescue of cholesterol storage phenotype in NPC1-deficient cells. (A, B) Confocal images of NPC1-mutant fibroblasts mock transfected (A) or transfected with RIDα (B) treated with dimethyl sulfoxide (DMSO) vehicle (left) or S58-035 (right) for 12 h and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (C) Quantification of peak LSO area per cell in cells treated similarly to cells in A and B as described in Materials and Methods . Data are presented as mean ± SEM (* p < 0.001). (D) Confocal image of NPC1-mutant fibroblasts transfected with RIDα treated with S58-035 for 12 h and stained with antibody to FLAG-RIDα and with BODIPY 493/503 and DAPI. Mock-transfected cell is shown in the same field as designated by an asterisk. (E, F) CT43 (E) and CT43-RIDα cells (F) treated with DMSO vehicle (left) or S58–035 (right) for 12 h and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (G) Quantification of peak LSO area per cell in cells treated similarly to cells in E and F as described in Materials and Methods . Data are presented as mean ± SEM (* p < 0.001). Boxed areas, regions of the image that were magnified. Bars, 10 μm. Nu, nucleus.

Journal: Molecular Biology of the Cell

Article Title: Adenovirus RIDα uncovers a novel pathway requiring ORP1L for lipid droplet formation independent of NPC1

doi: 10.1091/mbc.E12-10-0760

Figure Lengend Snippet: Inhibition of ACAT blocks RIDα rescue of cholesterol storage phenotype in NPC1-deficient cells. (A, B) Confocal images of NPC1-mutant fibroblasts mock transfected (A) or transfected with RIDα (B) treated with dimethyl sulfoxide (DMSO) vehicle (left) or S58-035 (right) for 12 h and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (C) Quantification of peak LSO area per cell in cells treated similarly to cells in A and B as described in Materials and Methods . Data are presented as mean ± SEM (* p < 0.001). (D) Confocal image of NPC1-mutant fibroblasts transfected with RIDα treated with S58-035 for 12 h and stained with antibody to FLAG-RIDα and with BODIPY 493/503 and DAPI. Mock-transfected cell is shown in the same field as designated by an asterisk. (E, F) CT43 (E) and CT43-RIDα cells (F) treated with DMSO vehicle (left) or S58–035 (right) for 12 h and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (G) Quantification of peak LSO area per cell in cells treated similarly to cells in E and F as described in Materials and Methods . Data are presented as mean ± SEM (* p < 0.001). Boxed areas, regions of the image that were magnified. Bars, 10 μm. Nu, nucleus.

Techniques: Inhibition, Mutagenesis, Transfection, Staining

RIDα does not modulate sterol-regulated gene expression in NPC1-deficient cells with stable RIDα expression. (A) Schematic summarizing homeostatic responses to increased free cholesterol (Ch). High levels of ER Ch become esterified by ACAT and also inhibit the SREBP pathway by facilitating SCAP–SREBP interactions and ER retention. Increased mitochondrial Ch stimulates 27-hydroxycholesterol (27-HC) biosynthesis, which binds Insig, contributing to SREBP ER retention, and activates nuclear LXR transcription factors. (B–E) HMGCR (B), LDLR (C), ABCA1 (D), and CYP7B (E) mRNA levels quantified by real-time PCR. Values are expressed as relative units after internal normalization to glyceraldehyde 3-phosphate dehydrogenase mRNA levels and compared with control samples from the same cell lines cultured in 10% FBS, which was set to 1 (dashed line) from three independent experiments. Data are presented as mean ± SEM. (F, G) Quantification of esterified cholesterol in Chinese hamster ovary, CT43, and CT43-RIDα cells (F) or shControl, shNPC1, and shNPC1-RIDα cells (G) with and without 24 h treatment with LDL using the Amplex Red Cholesterol Assay kit as described in Materials and Methods . Values were normalized to total cellular protein and are displayed as mean ± SEM. (H) Equal aliquots of total cellular protein from CT43 and CT43-RIDα cells or shNPC1 and shNPC1-RIDα cells were immunoblotted with antibodies to FLAG or actin for loading control.

Journal: Molecular Biology of the Cell

Article Title: Adenovirus RIDα uncovers a novel pathway requiring ORP1L for lipid droplet formation independent of NPC1

doi: 10.1091/mbc.E12-10-0760

Figure Lengend Snippet: RIDα does not modulate sterol-regulated gene expression in NPC1-deficient cells with stable RIDα expression. (A) Schematic summarizing homeostatic responses to increased free cholesterol (Ch). High levels of ER Ch become esterified by ACAT and also inhibit the SREBP pathway by facilitating SCAP–SREBP interactions and ER retention. Increased mitochondrial Ch stimulates 27-hydroxycholesterol (27-HC) biosynthesis, which binds Insig, contributing to SREBP ER retention, and activates nuclear LXR transcription factors. (B–E) HMGCR (B), LDLR (C), ABCA1 (D), and CYP7B (E) mRNA levels quantified by real-time PCR. Values are expressed as relative units after internal normalization to glyceraldehyde 3-phosphate dehydrogenase mRNA levels and compared with control samples from the same cell lines cultured in 10% FBS, which was set to 1 (dashed line) from three independent experiments. Data are presented as mean ± SEM. (F, G) Quantification of esterified cholesterol in Chinese hamster ovary, CT43, and CT43-RIDα cells (F) or shControl, shNPC1, and shNPC1-RIDα cells (G) with and without 24 h treatment with LDL using the Amplex Red Cholesterol Assay kit as described in Materials and Methods . Values were normalized to total cellular protein and are displayed as mean ± SEM. (H) Equal aliquots of total cellular protein from CT43 and CT43-RIDα cells or shNPC1 and shNPC1-RIDα cells were immunoblotted with antibodies to FLAG or actin for loading control.

Techniques: Gene Expression, Expressing, Real-time Polymerase Chain Reaction, Control, Cell Culture, Amplex Red Cholesterol Assay

RIDα does not rescue ER retention of defective NPC1 I1061T mutant protein in patient fibroblasts. (A–C) Confocal images of normal (A), mock-transfected homozygous NPC1 I1061T mutant (B), or homozygous NPC1 I1061T mutant fibroblasts transfected with FLAG-RIDα (C) and stained with antibodies to NPC1 and FLAG-RIDα and costained with antibodies to calreticulin (left) or LAMP1 (right). (D, E) Confocal images of homozygous NPC1 I1061T mutant fibroblasts mock transfected (D, left) or transfected with FLAG-RIDα (D, right; E) and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin (D) or with antibody to FLAG-RIDα and with BODIPY 493/503 and DAPI (E). Mock-transfected cells in E are shown in the same field and designated with an asterisk Boxed areas, regions of the image that were magnified. Bars, 10 μm. Nu, nucleus.

Journal: Molecular Biology of the Cell

Article Title: Adenovirus RIDα uncovers a novel pathway requiring ORP1L for lipid droplet formation independent of NPC1

doi: 10.1091/mbc.E12-10-0760

Figure Lengend Snippet: RIDα does not rescue ER retention of defective NPC1 I1061T mutant protein in patient fibroblasts. (A–C) Confocal images of normal (A), mock-transfected homozygous NPC1 I1061T mutant (B), or homozygous NPC1 I1061T mutant fibroblasts transfected with FLAG-RIDα (C) and stained with antibodies to NPC1 and FLAG-RIDα and costained with antibodies to calreticulin (left) or LAMP1 (right). (D, E) Confocal images of homozygous NPC1 I1061T mutant fibroblasts mock transfected (D, left) or transfected with FLAG-RIDα (D, right; E) and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin (D) or with antibody to FLAG-RIDα and with BODIPY 493/503 and DAPI (E). Mock-transfected cells in E are shown in the same field and designated with an asterisk Boxed areas, regions of the image that were magnified. Bars, 10 μm. Nu, nucleus.

Techniques: Mutagenesis, Transfection, Staining

siRNA knockdown of ORP1L blocks RIDα rescue of cholesterol storage phenotype in NPC1-deficient cells. (A) Schematic representation of ORP1L. ORP1L has amino-terminal ankyrin repeats, pleckstrin homology domain (PH), FFAT motif, and carboxy-terminal oxysterol binding-related domain (ORD; Suchanek et al. , 2007 ). GTP-Rab7 binding has been mapped to the ankyrin repeats ( Johansson et al. , 2005 ), and RIDα binds the ORD ( Shah et al. , 2007 ). (B) shControl cells were transfected with Cntl siRNA or shControl, shNPC1, or shNPC1-RIDα cells were transfected with ORP1L siRNA targeting the ORF, and equal aliquots of total cellular protein were immunoblotted with antibodies to ORP1L or actin for loading control. (C–E) Confocal images of shControl (C), shNPC1 (D), or shNPC1-RIDα cells (E) transfected with Cntl siRNA (left) or ORP1L siRNA targeting the ORF (right) and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (F) Quantification of peak LSO area per cell in cells treated similarly to cells in C–E as described in Materials and Methods . Data are presented as mean ± SEM (* p < 0.001). (G–I) Confocal images of shControl (G), shNPC1 (H), or shNPC1-RIDα cells (I) transfected with Cntl siRNA (left) or ORP1L siRNA (right) and stained with antibody to FLAG-RIDα and with BODIPY 493/503 and DAPI. (J) Quantification of esterified cholesterol in shControl, shNPC1, and shNPC1-RIDα cells transfected with Cntl or ORP1L siRNA using the Amplex Red Cholesterol Assay kit as described in Materials and Methods . Values were normalized to total cellular protein and are displayed as mean ± SEM (* p < 0.001). Boxed areas, regions of the image that were magnified. Bars, 10 μm. Nu, nucleus.

Journal: Molecular Biology of the Cell

Article Title: Adenovirus RIDα uncovers a novel pathway requiring ORP1L for lipid droplet formation independent of NPC1

doi: 10.1091/mbc.E12-10-0760

Figure Lengend Snippet: siRNA knockdown of ORP1L blocks RIDα rescue of cholesterol storage phenotype in NPC1-deficient cells. (A) Schematic representation of ORP1L. ORP1L has amino-terminal ankyrin repeats, pleckstrin homology domain (PH), FFAT motif, and carboxy-terminal oxysterol binding-related domain (ORD; Suchanek et al. , 2007 ). GTP-Rab7 binding has been mapped to the ankyrin repeats ( Johansson et al. , 2005 ), and RIDα binds the ORD ( Shah et al. , 2007 ). (B) shControl cells were transfected with Cntl siRNA or shControl, shNPC1, or shNPC1-RIDα cells were transfected with ORP1L siRNA targeting the ORF, and equal aliquots of total cellular protein were immunoblotted with antibodies to ORP1L or actin for loading control. (C–E) Confocal images of shControl (C), shNPC1 (D), or shNPC1-RIDα cells (E) transfected with Cntl siRNA (left) or ORP1L siRNA targeting the ORF (right) and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (F) Quantification of peak LSO area per cell in cells treated similarly to cells in C–E as described in Materials and Methods . Data are presented as mean ± SEM (* p < 0.001). (G–I) Confocal images of shControl (G), shNPC1 (H), or shNPC1-RIDα cells (I) transfected with Cntl siRNA (left) or ORP1L siRNA (right) and stained with antibody to FLAG-RIDα and with BODIPY 493/503 and DAPI. (J) Quantification of esterified cholesterol in shControl, shNPC1, and shNPC1-RIDα cells transfected with Cntl or ORP1L siRNA using the Amplex Red Cholesterol Assay kit as described in Materials and Methods . Values were normalized to total cellular protein and are displayed as mean ± SEM (* p < 0.001). Boxed areas, regions of the image that were magnified. Bars, 10 μm. Nu, nucleus.

Techniques: Knockdown, Binding Assay, Transfection, Control, Staining, Amplex Red Cholesterol Assay

Overexpression of ORP1L after siRNA knockdown restores the ability of RIDα to rescue the cholesterol storage phenotype in NPC1-deficient cells. (A) shNPC1-RIDα cells were transfected with Cntl siRNA or ORP1L siRNA targeting the 3′ UTR or cotransfected with ORP1L siRNA targeting the 3′ UTR along with wild-type or Δ560–563 GFP-ORP1L, and equal aliquots of total cellular protein were immunoblotted with antibodies to ORP1L, GFP, or actin for loading control. (B–E) Confocal images of shNPC1-RIDα cells transfected with Cntl siRNA (B), ORP1L siRNA targeting the 3′ UTR (C), or cotransfected with ORP1L siRNA targeting the 3′ UTR and wild-type (D) or Δ560–563 GFP-ORP1L (E) and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (F–I) Confocal images of shNPC1-RIDα cells transfected with Cntl siRNA (F), ORP1L siRNA targeting the 3′ UTR (G), or cotransfected with ORP1L siRNA targeting the 3′ UTR and wild-type (H) or Δ560–563 GFP-ORP1L (I) and stained with antibody to FLAG-RIDα and with LipidTOX deep red. (J) Quantification of esterified cholesterol in shNPC1-RIDα cells transfected with Cntl siRNA or ORP1L siRNA targeting the 3′ UTR or cotransfected with ORP1L siRNA targeting the 3′ UTR along with wild-type or Δ560–563 GFP-ORP1L using the Amplex Red Cholesterol Assay kit as described in Materials and Methods . Values were normalized to total cellular protein and are displayed as mean ± SEM (* p < 0.001). Boxed areas, regions of the image that were magnified. Bars, 10 μm. Nu, nucleus.

Journal: Molecular Biology of the Cell

Article Title: Adenovirus RIDα uncovers a novel pathway requiring ORP1L for lipid droplet formation independent of NPC1

doi: 10.1091/mbc.E12-10-0760

Figure Lengend Snippet: Overexpression of ORP1L after siRNA knockdown restores the ability of RIDα to rescue the cholesterol storage phenotype in NPC1-deficient cells. (A) shNPC1-RIDα cells were transfected with Cntl siRNA or ORP1L siRNA targeting the 3′ UTR or cotransfected with ORP1L siRNA targeting the 3′ UTR along with wild-type or Δ560–563 GFP-ORP1L, and equal aliquots of total cellular protein were immunoblotted with antibodies to ORP1L, GFP, or actin for loading control. (B–E) Confocal images of shNPC1-RIDα cells transfected with Cntl siRNA (B), ORP1L siRNA targeting the 3′ UTR (C), or cotransfected with ORP1L siRNA targeting the 3′ UTR and wild-type (D) or Δ560–563 GFP-ORP1L (E) and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (F–I) Confocal images of shNPC1-RIDα cells transfected with Cntl siRNA (F), ORP1L siRNA targeting the 3′ UTR (G), or cotransfected with ORP1L siRNA targeting the 3′ UTR and wild-type (H) or Δ560–563 GFP-ORP1L (I) and stained with antibody to FLAG-RIDα and with LipidTOX deep red. (J) Quantification of esterified cholesterol in shNPC1-RIDα cells transfected with Cntl siRNA or ORP1L siRNA targeting the 3′ UTR or cotransfected with ORP1L siRNA targeting the 3′ UTR along with wild-type or Δ560–563 GFP-ORP1L using the Amplex Red Cholesterol Assay kit as described in Materials and Methods . Values were normalized to total cellular protein and are displayed as mean ± SEM (* p < 0.001). Boxed areas, regions of the image that were magnified. Bars, 10 μm. Nu, nucleus.

Techniques: Over Expression, Knockdown, Transfection, Control, Staining, Amplex Red Cholesterol Assay

RIDα induces formation of LDs during Ad infection of NPC1-deficient cells. (A– C) Confocal images of shControl (A), shNPC1 (B), or shNPC2 cells (C) mock infected or infected with RIDα-overexpressing in 724 (middle) or RIDα-null dl 753 (right) Ad2. Cells were stained with BODIPY 493/503 and DAPI. Bars, 10 μm.

Journal: Molecular Biology of the Cell

Article Title: Adenovirus RIDα uncovers a novel pathway requiring ORP1L for lipid droplet formation independent of NPC1

doi: 10.1091/mbc.E12-10-0760

Figure Lengend Snippet: RIDα induces formation of LDs during Ad infection of NPC1-deficient cells. (A– C) Confocal images of shControl (A), shNPC1 (B), or shNPC2 cells (C) mock infected or infected with RIDα-overexpressing in 724 (middle) or RIDα-null dl 753 (right) Ad2. Cells were stained with BODIPY 493/503 and DAPI. Bars, 10 μm.

Techniques: Infection, Staining

Model depicting hypothetical mechanisms of RIDα/ORP1L-mediated transport of cholesterol from endosome to ER in NPC1-deficient cells. Cholesterol may be trafficked from endosomes to the ER by vesicular or nonvesicular mechanisms, including membrane contact sites, where close apposition of donor and acceptor membranes supports transport down a concentration gradient (A), vesicular transport, where cholesterol is packaged in transport vesicles to be delivered to the ER (B), or by transport proteins that bind cholesterol in a hydrophobic pocket protected from the cytosol and transport it to the ER (C). RIDα mediates the transport of cholesterol to the ER independent of NPC1 but dependent on NPC2 and ORP1L. Once delivered, cholesterol is acted upon by ACAT for incorporation into LDs and does not affect SREBP-regulated gene expression. CE, cholesterol ester; Ch, cholesterol.

Journal: Molecular Biology of the Cell

Article Title: Adenovirus RIDα uncovers a novel pathway requiring ORP1L for lipid droplet formation independent of NPC1

doi: 10.1091/mbc.E12-10-0760

Figure Lengend Snippet: Model depicting hypothetical mechanisms of RIDα/ORP1L-mediated transport of cholesterol from endosome to ER in NPC1-deficient cells. Cholesterol may be trafficked from endosomes to the ER by vesicular or nonvesicular mechanisms, including membrane contact sites, where close apposition of donor and acceptor membranes supports transport down a concentration gradient (A), vesicular transport, where cholesterol is packaged in transport vesicles to be delivered to the ER (B), or by transport proteins that bind cholesterol in a hydrophobic pocket protected from the cytosol and transport it to the ER (C). RIDα mediates the transport of cholesterol to the ER independent of NPC1 but dependent on NPC2 and ORP1L. Once delivered, cholesterol is acted upon by ACAT for incorporation into LDs and does not affect SREBP-regulated gene expression. CE, cholesterol ester; Ch, cholesterol.

Techniques: Membrane, Concentration Assay, Gene Expression

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