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Addgene inc gcwt n370s l444p vsvg pcdna3 1 constructs
Gcwt N370s L444p Vsvg Pcdna3 1 Constructs, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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gcwt n370s l444p vsvg pcdna3 1 constructs - by Bioz Stars, 2026-06

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A ELISA of 20 purified Nbs using wild-type GCase or N370S GCase coated on the bottom of the ELISA wells. An irrelevant Nb is used as negative control, while the positive control displays the signal of a Nb (Nb17) directly coated in the ELISA plate. Each ELISA signal is the result of three independent experiments represented as mean values (bars) with standard deviations (error bars). B Representative Bio-Layer interferometry (BLI) traces for binding of Nb1 to wild-type GCase (upper panel), and fitting of the signal amplitudes (Req) versus Nb1 concentration curve on the Langmuir equation to determine the K D values (lower panel). C Equilibrium dissociation constants (K D ) of the Nbs for wild-type GCase or N370S GCase. For wild-type GCase, BLI experiments were performed with biotinylated GCase immobilized on the Streptavidin sensors (see Supplementary Fig. for results of the experiment using the inverse set-up). For N370S GCase, C-terminally biotinylated Nbs were immobilized on the Streptavidin sensors. K D values are determined similar to ( B ). K D values for wild-type GCase are represented as mean values ± standard deviations ( n = 3), while reported K D values for N370S GCase are the result of a single experiment. Source data are deposited as Source Data files on Zenodo.

Journal: Nature Communications

Article Title: Developing nanobodies as allosteric molecular chaperones of glucocerebrosidase function

doi: 10.1038/s41467-025-60134-4

Figure Lengend Snippet: A ELISA of 20 purified Nbs using wild-type GCase or N370S GCase coated on the bottom of the ELISA wells. An irrelevant Nb is used as negative control, while the positive control displays the signal of a Nb (Nb17) directly coated in the ELISA plate. Each ELISA signal is the result of three independent experiments represented as mean values (bars) with standard deviations (error bars). B Representative Bio-Layer interferometry (BLI) traces for binding of Nb1 to wild-type GCase (upper panel), and fitting of the signal amplitudes (Req) versus Nb1 concentration curve on the Langmuir equation to determine the K D values (lower panel). C Equilibrium dissociation constants (K D ) of the Nbs for wild-type GCase or N370S GCase. For wild-type GCase, BLI experiments were performed with biotinylated GCase immobilized on the Streptavidin sensors (see Supplementary Fig. for results of the experiment using the inverse set-up). For N370S GCase, C-terminally biotinylated Nbs were immobilized on the Streptavidin sensors. K D values are determined similar to ( B ). K D values for wild-type GCase are represented as mean values ± standard deviations ( n = 3), while reported K D values for N370S GCase are the result of a single experiment. Source data are deposited as Source Data files on Zenodo.

Article Snippet: Gba1 −/− h N370S mice were purchased from the Jackson Laboratories ( https://www.jax.org/strain/032791 ).

Techniques: Enzyme-linked Immunosorbent Assay, Purification, Negative Control, Positive Control, Binding Assay, Concentration Assay

A Nb10 and Nb16 are able to significantly improve in vitro N370S GCase enzymatic activity. Isofagomine (IFG) was used as a negative control ( n = 6 replicates in three independent experiments, data represented as mean ± SEM, statistical analysis was performed using an Ordinary One Way Anova multiple comparison test); B GCase activity assay in gut lysates from h N370S GBA1 −/− mice incubated with Nb1, Nb4, Nb9, Nb10, and Nb16 showed that Nb10 and Nb16 are able to significantly improve in vitro N370S GCase enzymatic activity. ( n = 4 tissue per genotype, each tested in 3 independent experiments in 3 technical replicates, data represented as histograms showing the average value for the technical replicates for each experiment in each biological sample, statistical analysis was performed using an Ordinary One Way Anova multiple comparison test, DF Nbs = 5, DF residual = 55, F value = 4.926). C Co-expression of the N370S GCase mutant and the ER-Nb4 and ER-Nb9 in GBA1 KD cells induced an increase in the lysosomal N370S GCase activity of about 60–70%, as compared to ER-Mock transfected cells, while no effects were observed for ER-Nb1 and ER-Nb16 ( n = 8 for ER-Mock, n = 4 for ER-Nb1, Nb4, Nb9, n = 5 for ER-Nb16 replicates in three independent experiments, data represented as violin plots, an Ordinary One Way Anova test with multiple comparisons, after Shapiro–Wilk Normality test was used for the statistical analysis Df Nbs = 4, Df Residual 24, F = 14.96). D Co-expression of N370S GCase mutant and the lysosomal-targeted Nb16 in GBA1 KD cells significantly increased the lysosomal N370S GCase activity, as compared to lysosomes-Mock transfected cells, while Nb1, Nb4, and Nb9 did not affect lysosomal N370S GCase activity when targeted to the lysosome ( n = 11 for lyso-Mock, n = 4 for lyso-Nb1, Nb4, Nb9, n = 5 for lyso Nb16 replicates in three independent experiments, data represented as violin plots, a Kruskal–Wallis test with multiple comparisons, after Shapiro–Wilk Normality test was used for the statistical analysis). Source data are deposited as Source Data files on Zenodo. GCase glucocerebrosidase, Nb nanobody, PFB-FDGlu 5-(Pentafluorobenzoylamino)Fluorescein Di-β-D-Glucopyranoside.

Journal: Nature Communications

Article Title: Developing nanobodies as allosteric molecular chaperones of glucocerebrosidase function

doi: 10.1038/s41467-025-60134-4

Figure Lengend Snippet: A Nb10 and Nb16 are able to significantly improve in vitro N370S GCase enzymatic activity. Isofagomine (IFG) was used as a negative control ( n = 6 replicates in three independent experiments, data represented as mean ± SEM, statistical analysis was performed using an Ordinary One Way Anova multiple comparison test); B GCase activity assay in gut lysates from h N370S GBA1 −/− mice incubated with Nb1, Nb4, Nb9, Nb10, and Nb16 showed that Nb10 and Nb16 are able to significantly improve in vitro N370S GCase enzymatic activity. ( n = 4 tissue per genotype, each tested in 3 independent experiments in 3 technical replicates, data represented as histograms showing the average value for the technical replicates for each experiment in each biological sample, statistical analysis was performed using an Ordinary One Way Anova multiple comparison test, DF Nbs = 5, DF residual = 55, F value = 4.926). C Co-expression of the N370S GCase mutant and the ER-Nb4 and ER-Nb9 in GBA1 KD cells induced an increase in the lysosomal N370S GCase activity of about 60–70%, as compared to ER-Mock transfected cells, while no effects were observed for ER-Nb1 and ER-Nb16 ( n = 8 for ER-Mock, n = 4 for ER-Nb1, Nb4, Nb9, n = 5 for ER-Nb16 replicates in three independent experiments, data represented as violin plots, an Ordinary One Way Anova test with multiple comparisons, after Shapiro–Wilk Normality test was used for the statistical analysis Df Nbs = 4, Df Residual 24, F = 14.96). D Co-expression of N370S GCase mutant and the lysosomal-targeted Nb16 in GBA1 KD cells significantly increased the lysosomal N370S GCase activity, as compared to lysosomes-Mock transfected cells, while Nb1, Nb4, and Nb9 did not affect lysosomal N370S GCase activity when targeted to the lysosome ( n = 11 for lyso-Mock, n = 4 for lyso-Nb1, Nb4, Nb9, n = 5 for lyso Nb16 replicates in three independent experiments, data represented as violin plots, a Kruskal–Wallis test with multiple comparisons, after Shapiro–Wilk Normality test was used for the statistical analysis). Source data are deposited as Source Data files on Zenodo. GCase glucocerebrosidase, Nb nanobody, PFB-FDGlu 5-(Pentafluorobenzoylamino)Fluorescein Di-β-D-Glucopyranoside.

Article Snippet: Gba1 −/− h N370S mice were purchased from the Jackson Laboratories ( https://www.jax.org/strain/032791 ).

Techniques: In Vitro, Activity Assay, Negative Control, Comparison, Incubation, Expressing, Mutagenesis, Transfection

Schematics of pegRNA design and PE tools for the introduction of the GBA (N370S) mutation in hPSCs The pegRNA is composed of a protospacer sequence that targets the region to edit, a primer binding site (PBS) and a reverse transcript (RT) template that includes the edit (in this case, N370S in GBA). The PE3 tools include a pegRNA, a PE enzyme (Cas9-H840A) fused to a reverse transcriptase, illustrated as a blue oval here) and an additional nicking sgRNA targeting the targeting the complementary DNA strand, located 61 bp downstream from the pegRNA nicking site.

Journal: STAR Protocols

Article Title: Protocol for the design, conduct, and evaluation of prime editing in human pluripotent stem cells

doi: 10.1016/j.xpro.2023.102583

Figure Lengend Snippet: Schematics of pegRNA design and PE tools for the introduction of the GBA (N370S) mutation in hPSCs The pegRNA is composed of a protospacer sequence that targets the region to edit, a primer binding site (PBS) and a reverse transcript (RT) template that includes the edit (in this case, N370S in GBA). The PE3 tools include a pegRNA, a PE enzyme (Cas9-H840A) fused to a reverse transcriptase, illustrated as a blue oval here) and an additional nicking sgRNA targeting the targeting the complementary DNA strand, located 61 bp downstream from the pegRNA nicking site.

Article Snippet: pegRNA-GBA-4 (GBA N370S correction) oligos , , .

Techniques: Mutagenesis, Sequencing, Binding Assay, Reverse Transcription

Plasmids cocktail for GBA induction and GBA correction using PE3+p53DD

Journal: STAR Protocols

Article Title: Protocol for the design, conduct, and evaluation of prime editing in human pluripotent stem cells

doi: 10.1016/j.xpro.2023.102583

Figure Lengend Snippet: Plasmids cocktail for GBA induction and GBA correction using PE3+p53DD

Article Snippet: pegRNA-GBA-4 (GBA N370S correction) oligos , , .

Techniques: Plasmid Preparation

PCR primer design for Miseq PCR primer design for GBA (c. 1226A > G, p. N370S) mutation induction and correction.

Journal: STAR Protocols

Article Title: Protocol for the design, conduct, and evaluation of prime editing in human pluripotent stem cells

doi: 10.1016/j.xpro.2023.102583

Figure Lengend Snippet: PCR primer design for Miseq PCR primer design for GBA (c. 1226A > G, p. N370S) mutation induction and correction.

Article Snippet: pegRNA-GBA-4 (GBA N370S correction) oligos , , .

Techniques: Mutagenesis

Miseq data analysis for a representative GBA (N370S) induction experiment The data was extracted from the CRISPResso2 website. (A) The number of reads input, after preprocessing, and after alignment to amplicons. We use this data to check the quality of the PCR product and the Miseq. (B) Alignment and editing frequency of unmodified and modified alleles of pegRNA. The modified alleles include the insertions, deletions, and substitutions at the nicking site of pegRNA (-3), which we use for checking indel frequency of the editing of pegRNA. (C) Alignment and editing frequency of unmodified and modified alleles of nicking sgRNA. The modified alleles include the insertions, deletions, and substitutions at the nicking site of nicking sgRNA (-3), which we use for checking indel frequency of induced nicking by sgRNA for PE3. (D) Visualization of the distribution of identified alleles around the pegRNA spacer. 31.05% of reads carried the A-to-G change at the desired GBA (N370) locus.

Journal: STAR Protocols

Article Title: Protocol for the design, conduct, and evaluation of prime editing in human pluripotent stem cells

doi: 10.1016/j.xpro.2023.102583

Figure Lengend Snippet: Miseq data analysis for a representative GBA (N370S) induction experiment The data was extracted from the CRISPResso2 website. (A) The number of reads input, after preprocessing, and after alignment to amplicons. We use this data to check the quality of the PCR product and the Miseq. (B) Alignment and editing frequency of unmodified and modified alleles of pegRNA. The modified alleles include the insertions, deletions, and substitutions at the nicking site of pegRNA (-3), which we use for checking indel frequency of the editing of pegRNA. (C) Alignment and editing frequency of unmodified and modified alleles of nicking sgRNA. The modified alleles include the insertions, deletions, and substitutions at the nicking site of nicking sgRNA (-3), which we use for checking indel frequency of induced nicking by sgRNA for PE3. (D) Visualization of the distribution of identified alleles around the pegRNA spacer. 31.05% of reads carried the A-to-G change at the desired GBA (N370) locus.

Article Snippet: pegRNA-GBA-4 (GBA N370S correction) oligos , , .

Techniques: Modification

Miseq data analysis for a representative GBA (N370S) correction experiment The data was extracted from the CRISPResso2 website. (A) The number of reads input, after preprocessing, and after alignment to amplicons. We use this data to check the quality of the PCR product and the Miseq. (B) Alignment and editing frequency of unmodified and modified alleles of pegRNA. The modified alleles include the insertions, deletions, and substitutions at the nicking site of pegRNA (-3), which we use for checking indel frequency of the editing of pegRNA. (C) Alignment and editing frequency of unmodified and modified alleles of nicking sgRNA. The modified alleles include the insertions, deletions, and substitutions at the nicking site of nicking sgRNA (-3), which we use for checking indel frequency of induced nicking by sgRNA for PE3. (D) Visualization of the distribution of identified alleles around the pegRNA spacer. Correction % = (63.20%–34.67%)/2 = 14.27%, which is the GBA correction frequency for the patient iPSC that carried a heterozygous GBA (N370S) mutation.

Journal: STAR Protocols

Article Title: Protocol for the design, conduct, and evaluation of prime editing in human pluripotent stem cells

doi: 10.1016/j.xpro.2023.102583

Figure Lengend Snippet: Miseq data analysis for a representative GBA (N370S) correction experiment The data was extracted from the CRISPResso2 website. (A) The number of reads input, after preprocessing, and after alignment to amplicons. We use this data to check the quality of the PCR product and the Miseq. (B) Alignment and editing frequency of unmodified and modified alleles of pegRNA. The modified alleles include the insertions, deletions, and substitutions at the nicking site of pegRNA (-3), which we use for checking indel frequency of the editing of pegRNA. (C) Alignment and editing frequency of unmodified and modified alleles of nicking sgRNA. The modified alleles include the insertions, deletions, and substitutions at the nicking site of nicking sgRNA (-3), which we use for checking indel frequency of induced nicking by sgRNA for PE3. (D) Visualization of the distribution of identified alleles around the pegRNA spacer. Correction % = (63.20%–34.67%)/2 = 14.27%, which is the GBA correction frequency for the patient iPSC that carried a heterozygous GBA (N370S) mutation.

Article Snippet: pegRNA-GBA-4 (GBA N370S correction) oligos , , .

Techniques: Modification, Mutagenesis

Journal: STAR Protocols

Article Title: Protocol for the design, conduct, and evaluation of prime editing in human pluripotent stem cells

doi: 10.1016/j.xpro.2023.102583

Figure Lengend Snippet:

Article Snippet: pegRNA-GBA-4 (GBA N370S correction) oligos , , .

Techniques: Recombinant, Lysis, Neutralization, Purification, Gel Extraction, Plasmid Preparation, Virus, Sequencing, Agarose Gel Electrophoresis

GCase protein level and activity in patient-derived fibroblasts. ( A ) Immunoblot and quantification for the GCase protein level in fibroblasts normalized to WT/WT control fibroblasts as shown graphically as mean with SEM ( * * P < 0.01, * * * P < 0.001, * * * * P < 0.001; n = 3). Three technical repeats. The n for each genotype per experiment was WT/WT n = 3; E326K/WT n = 1; E326K/E326K n = 1; L444P/L444P n = 2; N370S/N370S n = 2. ( B ) Immunoblot and quantification for the GCase protein level in adult and young control fibroblasts normalized to WT/WT control fibroblasts ( * * * * P < 0.0001; n = 3). Three technical repeats. ( C ) The expression of GBA mRNA levels in patient fibroblasts was quantified and normalized to WT/WT controls. For each experiment, two biological replicates were used for each cell line. For quantification, GBA expression for each cell line was calculated, pooled and averaged for each genotype. Three technical repeats. The n for each genotype per experiment was WT/WT n = 3; E326K/WT n = 1; E326K/E326K n = 1; L444P/L444P n = 2; N370S/N370S n = 2. Graphs show the mean and error bars show the SEM. GCase activity assay with M-Glu was performed at ( D ) pH 5.4 with NaT and ( E ) pH 4.5 on fibroblast cell lysates. All data normalized to WT/WT control fibroblasts. GCase activity in in adult and young control fibroblasts at ( F ) pH 5.4 with NaT and (G) pH 4.5. ( * * P < 0.01, * * * P < 0.001, * * * * P < 0.0001; n = 4; ns, not significant). All data normalized to WT/WT control fibroblasts. Three technical repeats. The n for each genotype per experiment was WT/WT n = 2; young WT/WT n = 2; L444P/L444P n = 2. All graphs show the mean with error bars as the SEM. Statistical test used was one-away ANOVA with Tukey’s post hoc analysis. Raw data can be found at: https://doi.org/10.5281/zenodo.6985167 .

Journal: Human Molecular Genetics

Article Title: The GBA variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in human cell lines

doi: 10.1093/hmg/ddac233

Figure Lengend Snippet: GCase protein level and activity in patient-derived fibroblasts. ( A ) Immunoblot and quantification for the GCase protein level in fibroblasts normalized to WT/WT control fibroblasts as shown graphically as mean with SEM ( * * P < 0.01, * * * P < 0.001, * * * * P < 0.001; n = 3). Three technical repeats. The n for each genotype per experiment was WT/WT n = 3; E326K/WT n = 1; E326K/E326K n = 1; L444P/L444P n = 2; N370S/N370S n = 2. ( B ) Immunoblot and quantification for the GCase protein level in adult and young control fibroblasts normalized to WT/WT control fibroblasts ( * * * * P < 0.0001; n = 3). Three technical repeats. ( C ) The expression of GBA mRNA levels in patient fibroblasts was quantified and normalized to WT/WT controls. For each experiment, two biological replicates were used for each cell line. For quantification, GBA expression for each cell line was calculated, pooled and averaged for each genotype. Three technical repeats. The n for each genotype per experiment was WT/WT n = 3; E326K/WT n = 1; E326K/E326K n = 1; L444P/L444P n = 2; N370S/N370S n = 2. Graphs show the mean and error bars show the SEM. GCase activity assay with M-Glu was performed at ( D ) pH 5.4 with NaT and ( E ) pH 4.5 on fibroblast cell lysates. All data normalized to WT/WT control fibroblasts. GCase activity in in adult and young control fibroblasts at ( F ) pH 5.4 with NaT and (G) pH 4.5. ( * * P < 0.01, * * * P < 0.001, * * * * P < 0.0001; n = 4; ns, not significant). All data normalized to WT/WT control fibroblasts. Three technical repeats. The n for each genotype per experiment was WT/WT n = 2; young WT/WT n = 2; L444P/L444P n = 2. All graphs show the mean with error bars as the SEM. Statistical test used was one-away ANOVA with Tukey’s post hoc analysis. Raw data can be found at: https://doi.org/10.5281/zenodo.6985167 .

Article Snippet: SH-SY5Y cells were transfected with a pcDNATM3.1 (+) mammalian expression vector (ThermoFisher, Cat#V79020) containing wild type (WT GBA pcDNA3.1; Addgene plasmid #188580; RRID: Addgene_188 580), E326K (E326K GBA pcDNA3.1; Addgene plasmid #188581; RRID: Addgene_188 581), L444P (L444P GBA pcDNA3.1; Addgene plasmid #188582; RRID: Addgene_188 582) or N370S (N370S GBA pcDNA3.1; Addgene plasmid #188583; RRID: Addgene_188 583), GBA cDNA.

Techniques: Activity Assay, Derivative Assay, Western Blot, Control, Expressing

Lysosomal content and function in patient-derived fibroblasts. ( A ) LAMP1 levels were measured via western blot and quantified to assess the overall endo-lysosomal content of the fibroblasts. Data quantified and normalized to WT/WT fibroblast controls. Three technical repeats. ( B ) LAMP1 levels measured via western blot and compared with young control fibroblasts. Lysosomal function in patient-derived fibroblasts. Three technical repeats. ( C ) β-Galactosidase and ( D ) β-hexosaminidase were measured at pH 4.1 to assess the overall lysosomal function in fibroblasts. All data normalized to WT/WT control fibroblasts. Four technical repeats. The activities of lysosomal hydrolases, ( E ) β-galactosidase and ( F ) β-hexosaminidase were measured with young control fibroblasts. Four technical repeats. The n for each genotype per experiment was WT/WT n = 3; E326K/WT n = 1; E326K/E326K n = 1; L444P/L444P n = 2; N370S/N370S n = 2. For the analysis of young controls, WT/WT n = 2; young WT/WT n = 2; L444P/L444P n = 2. All graphs show the mean with error bars as the SEM. The statistical test used was one-away ANOVA with Tukey’s post hoc analysis. Raw data can be found at https://doi.org/10.5281/zenodo.6985167 .

Journal: Human Molecular Genetics

Article Title: The GBA variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in human cell lines

doi: 10.1093/hmg/ddac233

Figure Lengend Snippet: Lysosomal content and function in patient-derived fibroblasts. ( A ) LAMP1 levels were measured via western blot and quantified to assess the overall endo-lysosomal content of the fibroblasts. Data quantified and normalized to WT/WT fibroblast controls. Three technical repeats. ( B ) LAMP1 levels measured via western blot and compared with young control fibroblasts. Lysosomal function in patient-derived fibroblasts. Three technical repeats. ( C ) β-Galactosidase and ( D ) β-hexosaminidase were measured at pH 4.1 to assess the overall lysosomal function in fibroblasts. All data normalized to WT/WT control fibroblasts. Four technical repeats. The activities of lysosomal hydrolases, ( E ) β-galactosidase and ( F ) β-hexosaminidase were measured with young control fibroblasts. Four technical repeats. The n for each genotype per experiment was WT/WT n = 3; E326K/WT n = 1; E326K/E326K n = 1; L444P/L444P n = 2; N370S/N370S n = 2. For the analysis of young controls, WT/WT n = 2; young WT/WT n = 2; L444P/L444P n = 2. All graphs show the mean with error bars as the SEM. The statistical test used was one-away ANOVA with Tukey’s post hoc analysis. Raw data can be found at https://doi.org/10.5281/zenodo.6985167 .

Techniques: Derivative Assay, Western Blot, Control

ER retention and ER stress in patient-derived fibroblasts. ( A ) Fibroblast cell lysates (20 μg of WT, E326K and N370S mutants and 70 μg of L444P mutants) were treated with or without endoglycosidase-H (Endo H) and GCase protein species analysed by western blot. WT/WT cell lysate (20 μg) was treated with Peptide-N-Glycosidase F (PNGase F) as a positive control. Figure shows blots at long and short exposures. The two normal species of GCase detected in fibroblasts are indicated by arrows. An additional lower molecular weight band was observed, indicating ER retained GCase, in L444P/L444P fibroblasts following endo-H treatment (black asterisk). The WT/WT cell line treated with PNGase exhibited a lower molecular weight band, indicating a GCase species with no glycosylation (Δ). ( B ) Quantitative analysis of Endo H digestion displayed as the density of bands for ER resident GCase divided by the density of bands for total GCase protein, normalized to the β-actin band density. Data normalized to WT/WT that was set at 1. Three technical repeats. The n for each genotype per experiment was WT/WT n = 2; E326K/WT n = 1; E326K/E326K n = 1; L444P/L444P n = 2; N370S/N370S n = 2. ER stress was analysed by ( C ) quantifying the BiP protein level in fibroblasts. Results are normalized to WT/WT control fibroblasts. Three technical repeats. ( D ) The expression of CHOP mRNA levels in patient fibroblasts was quantified and normalized to WT/WT controls. For each experiment, two biological replicates were used for each cell line. For quantification, CHOP expression for each cell line was calculated, pooled and averaged for each genotype. Three technical repeats. The n for each genotype per experiment was WT/WT n = 3; E326K/WT n = 1; E326K/E326K n = 1; L444P/L444P n = 2; N370S/N370S n = 2. Graphs show the mean with error bars showing the SEM. The statistical test used was one-way ANOVA with Tukey post hoc analysis ( * P < 0.05, * * P < 0.01; n = 3). Raw data can be found at https://doi.org/10.5281/zenodo.6985167 .

Journal: Human Molecular Genetics

Article Title: The GBA variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in human cell lines

doi: 10.1093/hmg/ddac233

Figure Lengend Snippet: ER retention and ER stress in patient-derived fibroblasts. ( A ) Fibroblast cell lysates (20 μg of WT, E326K and N370S mutants and 70 μg of L444P mutants) were treated with or without endoglycosidase-H (Endo H) and GCase protein species analysed by western blot. WT/WT cell lysate (20 μg) was treated with Peptide-N-Glycosidase F (PNGase F) as a positive control. Figure shows blots at long and short exposures. The two normal species of GCase detected in fibroblasts are indicated by arrows. An additional lower molecular weight band was observed, indicating ER retained GCase, in L444P/L444P fibroblasts following endo-H treatment (black asterisk). The WT/WT cell line treated with PNGase exhibited a lower molecular weight band, indicating a GCase species with no glycosylation (Δ). ( B ) Quantitative analysis of Endo H digestion displayed as the density of bands for ER resident GCase divided by the density of bands for total GCase protein, normalized to the β-actin band density. Data normalized to WT/WT that was set at 1. Three technical repeats. The n for each genotype per experiment was WT/WT n = 2; E326K/WT n = 1; E326K/E326K n = 1; L444P/L444P n = 2; N370S/N370S n = 2. ER stress was analysed by ( C ) quantifying the BiP protein level in fibroblasts. Results are normalized to WT/WT control fibroblasts. Three technical repeats. ( D ) The expression of CHOP mRNA levels in patient fibroblasts was quantified and normalized to WT/WT controls. For each experiment, two biological replicates were used for each cell line. For quantification, CHOP expression for each cell line was calculated, pooled and averaged for each genotype. Three technical repeats. The n for each genotype per experiment was WT/WT n = 3; E326K/WT n = 1; E326K/E326K n = 1; L444P/L444P n = 2; N370S/N370S n = 2. Graphs show the mean with error bars showing the SEM. The statistical test used was one-way ANOVA with Tukey post hoc analysis ( * P < 0.05, * * P < 0.01; n = 3). Raw data can be found at https://doi.org/10.5281/zenodo.6985167 .

Techniques: Derivative Assay, Western Blot, Positive Control, Molecular Weight, Glycoproteomics, Control, Expressing

GBA levels, lysosomal function and ER stress in SH-SY5Y cells overexpressing mutant GCase. ( A ) Quantification of GBA mRNA levels in stable SH-SY5Y cell lines, normalized to untransfected SH-SY5Y cells (set at 1). Three technical repeats. The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1. ( B ) Immunoblot and ( C ) quantification of GBA protein levels in stable SH-SY5Y cell lines. Data normalized to untransfected SH-SY5Y cells (set at 1). Graphs show the mean with SEM. The statistical test used was one-way ANOVA with Tukey’s post hoc analysis ( * P < 0.05, * * P < 0.01, * * * P < 0.001, * * * * P < 0.0001; n = 4). Four technical repeats. ( D ) GCase activity in nmole/hr/mg in stable SH-SY5Y cell lines measured at pH 5.4 with NaT and normalized to untransfected SH-SY5Y cells (set at 1) Graphs show the mean with SEM. The statistical test used was one-way ANOVA with Tukey’s post hoc analysis ( * P < 0.05, * * P < 0.01, * * * P < 0.001; n = 4). The activities of lysosomal hydrolases, β-galactosidase and β-hexosaminidase were measured at pH 5.1 in SH-SY5Y stable cell lines. Four technical repeats. ( E ) β-galactosidase activity in nmole/0.5 h/mg in undifferentiated SH-SY5Y clones normalized to untransfected SH-SY5Y cells. ( F ) β-Hexosaminidase activity in nmole/0.5 h/mg measured in undifferentiated SH-SY5Y clones normalized to untransfected SH-SY5Y cells. Four technical repeats. ( G ) LAMP1 levels were measured via western blot and ( H ) quantified to assess the overall endo-lysosomal content of the SH-SY5Y cell lines. Immunoblot and quantification for LAMP1 protein level in SH-SY5Y stable cell lines. Data normalized to wild-type SH-SY5Y cells. Four technical repeats. ( I ) Immunoblot and ( J ) quantification of BiP protein level in SH-SY5Y clones. Data normalized to untransfected SH-SY5Y cells. Six technical repeats. The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1; GFP n = 1. Graphs show the mean with SEM. The statistical test used was one-way ANOVA with Tukey’s post hoc analysis( * P < 0.05. * * P < 0.01; n = 3). Raw data can be found at https://doi.org/10.5281/zenodo.6985167 .

Journal: Human Molecular Genetics

Article Title: The GBA variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in human cell lines

doi: 10.1093/hmg/ddac233

Figure Lengend Snippet: GBA levels, lysosomal function and ER stress in SH-SY5Y cells overexpressing mutant GCase. ( A ) Quantification of GBA mRNA levels in stable SH-SY5Y cell lines, normalized to untransfected SH-SY5Y cells (set at 1). Three technical repeats. The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1. ( B ) Immunoblot and ( C ) quantification of GBA protein levels in stable SH-SY5Y cell lines. Data normalized to untransfected SH-SY5Y cells (set at 1). Graphs show the mean with SEM. The statistical test used was one-way ANOVA with Tukey’s post hoc analysis ( * P < 0.05, * * P < 0.01, * * * P < 0.001, * * * * P < 0.0001; n = 4). Four technical repeats. ( D ) GCase activity in nmole/hr/mg in stable SH-SY5Y cell lines measured at pH 5.4 with NaT and normalized to untransfected SH-SY5Y cells (set at 1) Graphs show the mean with SEM. The statistical test used was one-way ANOVA with Tukey’s post hoc analysis ( * P < 0.05, * * P < 0.01, * * * P < 0.001; n = 4). The activities of lysosomal hydrolases, β-galactosidase and β-hexosaminidase were measured at pH 5.1 in SH-SY5Y stable cell lines. Four technical repeats. ( E ) β-galactosidase activity in nmole/0.5 h/mg in undifferentiated SH-SY5Y clones normalized to untransfected SH-SY5Y cells. ( F ) β-Hexosaminidase activity in nmole/0.5 h/mg measured in undifferentiated SH-SY5Y clones normalized to untransfected SH-SY5Y cells. Four technical repeats. ( G ) LAMP1 levels were measured via western blot and ( H ) quantified to assess the overall endo-lysosomal content of the SH-SY5Y cell lines. Immunoblot and quantification for LAMP1 protein level in SH-SY5Y stable cell lines. Data normalized to wild-type SH-SY5Y cells. Four technical repeats. ( I ) Immunoblot and ( J ) quantification of BiP protein level in SH-SY5Y clones. Data normalized to untransfected SH-SY5Y cells. Six technical repeats. The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1; GFP n = 1. Graphs show the mean with SEM. The statistical test used was one-way ANOVA with Tukey’s post hoc analysis( * P < 0.05. * * P < 0.01; n = 3). Raw data can be found at https://doi.org/10.5281/zenodo.6985167 .

Techniques: Mutagenesis, Western Blot, Activity Assay, Stable Transfection, Clone Assay

$Soluble and insoluble alpha-synuclein levels in SH-SY5Y stable cell lines expressing mutant GBA. ( A ) Immunoblot of alpha-synuclein protein level in SH-SY5Y clones. ( B ) Quantification of alpha-synuclein immunoblotting in SH-SY5Y clones normalized to wild-type clones. The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1; GFP n = 1. Six technical repeats. ( C ) Quantification of SNCA mRNA levels in SH-SY5Y stable clones normalized to wild-type clones ( n = 3). The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1; GFP n = 1. Two biological repeats and six technical repeats. TX-100 soluble and insoluble fractions (urea-SDS) were made from cells and analysed for alpha-synuclein by western blotting. HMW alpha-synuclein species (arrow) were detected in urea-SDS fractions. Monomeric alpha-synuclein was present in the TX-100 soluble fraction and some urea-SDS fractions. ( D ) Immunoblot for all SH-SY5Y cell lines over-expressing mutant GBA protein. Appropriate control cell lines were also analysed: ( E ) HAP1 alpha-synuclein knockout cells (KO), ( F ) untransfected SH-SY5Y cells (UT) and ( G ) GFP over-expressing SH-SY5Y cells (GFP). ( H ) Quantification of soluble and insoluble alpha-synuclein immunoblots. Data normalized to wild-type clones. The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1; GFP n = 1. Ten technical repeats. Graphs show the mean with SEM. The statistical test used was one-way ANOVA with Tukey’s post hoc analysis or two-way ANOVA with Tukey’s post hoc analysis ( * P < 0.05; n = 16). Raw data can be found at https://doi.org/10.5281/zenodo.6985167 .$

Journal: Human Molecular Genetics

Article Title: The GBA variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in human cell lines

doi: 10.1093/hmg/ddac233

Figure Lengend Snippet: Soluble and insoluble alpha-synuclein levels in SH-SY5Y stable cell lines expressing mutant GBA. ( A ) Immunoblot of alpha-synuclein protein level in SH-SY5Y clones. ( B ) Quantification of alpha-synuclein immunoblotting in SH-SY5Y clones normalized to wild-type clones. The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1; GFP n = 1. Six technical repeats. ( C ) Quantification of SNCA mRNA levels in SH-SY5Y stable clones normalized to wild-type clones ( n = 3). The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1; GFP n = 1. Two biological repeats and six technical repeats. TX-100 soluble and insoluble fractions (urea-SDS) were made from cells and analysed for alpha-synuclein by western blotting. HMW alpha-synuclein species (arrow) were detected in urea-SDS fractions. Monomeric alpha-synuclein was present in the TX-100 soluble fraction and some urea-SDS fractions. ( D ) Immunoblot for all SH-SY5Y cell lines over-expressing mutant GBA protein. Appropriate control cell lines were also analysed: ( E ) HAP1 alpha-synuclein knockout cells (KO), ( F ) untransfected SH-SY5Y cells (UT) and ( G ) GFP over-expressing SH-SY5Y cells (GFP). ( H ) Quantification of soluble and insoluble alpha-synuclein immunoblots. Data normalized to wild-type clones. The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1; GFP n = 1. Ten technical repeats. Graphs show the mean with SEM. The statistical test used was one-way ANOVA with Tukey’s post hoc analysis or two-way ANOVA with Tukey’s post hoc analysis ( * P < 0.05; n = 16). Raw data can be found at https://doi.org/10.5281/zenodo.6985167 .

Techniques: Stable Transfection, Expressing, Mutagenesis, Western Blot, Clone Assay, Control, Knock-Out

Lipid droplets in GBA mutant cells. ( A ) Fibroblast lines harbouring the E326K mutations were starved in Opti-MEM overnight and incubated with and without 100 μM OA for 5 h. Cells were stained with lipophilic fluorescent probe BODIPY 493/503. Lipid droplets represent punctate structures. Three representative images from each genotype shown (−) denotes untreated and (+) denotes treated with OA. All cells were counted in the images and the number of lipid droplets counted and normalized to cell area using ImageJ. Scale bar represents 20 μm. The n for each genotype per experiment was WT/WT n = 6; E326K/WT n = 1; E326K/E326K n = 1; >100 cells analysed per genotype. Quantification of lipid droplets in ( B ) control and E326K/+ fibroblasts and ( C) control and E326K/E326K fibroblasts displayed as the mean with error bars showing SEM. The statistical test used was one-way ANOVA with Tukey post hoc analysis or two-way ANOVA with Tukey’s post hoc analysis ( * * * * P < 0.0001). ( D ) SH-SY5Y cells over-expressing mutant GCase protein were grown on coverslips and stained with lipophilic fluorescent probe BODIPY 493/503. Lipid droplets represent punctate structures. Three representative images from each genotype shown. All cells were counted in the images and the number of lipid droplets counted and normalized to cell area using ImageJ. The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1; GFP n = 1. > 50 cells analysed per genotype. ( E ) Quantification of lipid droplets per cell area shown as the mean with SEM. The statistical test used was one-way ANOVA with Tukey post hoc analysis or two-way ANOVA with Tukey’s post hoc analysis ( * * P < 0.01; * * * * P < 0.0001). ( F ) SH-SY5Y cells over-expressing mutant GCase protein were starved in Opti-MEM overnight and incubated with and without 100 μM OA for 5 h. Cells were stained with lipophilic fluorescent probe BODIPY 493/503. Lipid droplets represent punctate structures. Three representative images from each genotype shown (−) denotes untreated and (+) denotes treated with OA. All cells were counted in the images and the number of lipid droplets counted and normalized to cell area using ImageJ. The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1; GFP n = 1. > 50 cells analysed per genotype. ( G ) Quantification of lipid droplets per cell area displayed as mean with error bars showing SEM. Statistical test used was one-way ANOVA with Tukey post hoc analysis or two-way ANOVA with Tukey’s post hoc analysis ( * P < 0.05; * * * * P < 0.0001). Scale bar represents 20 μm. Raw data can be found at https://doi.org/10.5281/zenodo.6985167 .

Journal: Human Molecular Genetics

Article Title: The GBA variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in human cell lines

doi: 10.1093/hmg/ddac233

Figure Lengend Snippet: Lipid droplets in GBA mutant cells. ( A ) Fibroblast lines harbouring the E326K mutations were starved in Opti-MEM overnight and incubated with and without 100 μM OA for 5 h. Cells were stained with lipophilic fluorescent probe BODIPY 493/503. Lipid droplets represent punctate structures. Three representative images from each genotype shown (−) denotes untreated and (+) denotes treated with OA. All cells were counted in the images and the number of lipid droplets counted and normalized to cell area using ImageJ. Scale bar represents 20 μm. The n for each genotype per experiment was WT/WT n = 6; E326K/WT n = 1; E326K/E326K n = 1; >100 cells analysed per genotype. Quantification of lipid droplets in ( B ) control and E326K/+ fibroblasts and ( C) control and E326K/E326K fibroblasts displayed as the mean with error bars showing SEM. The statistical test used was one-way ANOVA with Tukey post hoc analysis or two-way ANOVA with Tukey’s post hoc analysis ( * * * * P < 0.0001). ( D ) SH-SY5Y cells over-expressing mutant GCase protein were grown on coverslips and stained with lipophilic fluorescent probe BODIPY 493/503. Lipid droplets represent punctate structures. Three representative images from each genotype shown. All cells were counted in the images and the number of lipid droplets counted and normalized to cell area using ImageJ. The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1; GFP n = 1. > 50 cells analysed per genotype. ( E ) Quantification of lipid droplets per cell area shown as the mean with SEM. The statistical test used was one-way ANOVA with Tukey post hoc analysis or two-way ANOVA with Tukey’s post hoc analysis ( * * P < 0.01; * * * * P < 0.0001). ( F ) SH-SY5Y cells over-expressing mutant GCase protein were starved in Opti-MEM overnight and incubated with and without 100 μM OA for 5 h. Cells were stained with lipophilic fluorescent probe BODIPY 493/503. Lipid droplets represent punctate structures. Three representative images from each genotype shown (−) denotes untreated and (+) denotes treated with OA. All cells were counted in the images and the number of lipid droplets counted and normalized to cell area using ImageJ. The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1; GFP n = 1. > 50 cells analysed per genotype. ( G ) Quantification of lipid droplets per cell area displayed as mean with error bars showing SEM. Statistical test used was one-way ANOVA with Tukey post hoc analysis or two-way ANOVA with Tukey’s post hoc analysis ( * P < 0.05; * * * * P < 0.0001). Scale bar represents 20 μm. Raw data can be found at https://doi.org/10.5281/zenodo.6985167 .

Techniques: Mutagenesis, Incubation, Staining, Control, Expressing

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