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ATCC human wildtype wt asyn

a Experimental setup for investigating FICD and <t>aSyn</t> interaction <t>in</t> <t>H4-Ctrl</t> and H4-aSyn cells transiently transfected with FICD-WT, catalytically inactive FICD-H363A, or constitutively active FICD-E234G. Mock transfection served as baseline. b . Venn diagram of AMPylated proteins detected in H4-aSyn cells with mock, FICD-WT or FICD-E234G transfection. Overexpression of FICD-E234G resulted in an increase in AMPylated proteins. c KEGG pathway enrichment of AMPylated proteins in FICD-WT and FICD-E234G-transfected H4-aSyn cells. The “lysosome” pathway reaches the highest statistical significance. Bar color indicates statistical significance expressed as -log10(FDR). d AMPylation enrichment in H4-aSyn cells overexpressing FICD-WT (left) and FICD-E234G (right). Volcano plot depicts log2 fold change (pro-N6pA versus DMSO) versus –log10 p-value; dashed lines mark significance cut-offs (p < 0.01, -log2(pro-N6pA/DMSO) > 1). Significantly enriched AMPylated proteins are highlighted in blue. Representative lysosomal proteins and aSyn (SNCA) are annotated (for c - d: n = 2; n = 2 technical replicates / experiment). e Whole proteomic analysis and hierarchical clustering of proteome profiles of H4-Ctrl and H4-aSyn cells, either transfected with mock, FICD-WT, FICD-H363A, or FICD-E234G. Columns represent experimental conditions, partitioned into four clusters ( – ), while rows correspond to differentially regulated proteins (clusters I-IV). On the right, top 3 over-represented KEGG pathways for each row cluster are shown. Z-score: normalized expression scale from lower (blue) to higher (red) levels. The “protein processing in endoplasmic reticulum” pathway is overrepresented in row clusters I and II. f Heatmap showing expression of a subset of 23 proteins within the KEEG “protein processing in endoplasmic reticulum” pathway that are upregulated upon FICD-E234G overexpression (For subset identification, refer to Fig. S3i, cluster 5). Proteins involved in the UPR are highlighted in bold (for e - f: n = 3).

Human Wildtype Wt Asyn, supplied by ATCC, used in various techniques. Bioz Stars score: 96/100, based on 453 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Aberrant FICD-mediated AMPylation drives α-Synuclein pathology and overall protein dyshomeostasis in dopaminergic neurons in Parkinson’s disease"

Article Title: Aberrant FICD-mediated AMPylation drives α-Synuclein pathology and overall protein dyshomeostasis in dopaminergic neurons in Parkinson’s disease

Journal: bioRxiv

doi: 10.64898/2026.03.30.715195

a Experimental setup for investigating FICD and aSyn interaction in H4-Ctrl and H4-aSyn cells transiently transfected with FICD-WT, catalytically inactive FICD-H363A, or constitutively active FICD-E234G. Mock transfection served as baseline. b . Venn diagram of AMPylated proteins detected in H4-aSyn cells with mock, FICD-WT or FICD-E234G transfection. Overexpression of FICD-E234G resulted in an increase in AMPylated proteins. c KEGG pathway enrichment of AMPylated proteins in FICD-WT and FICD-E234G-transfected H4-aSyn cells. The “lysosome” pathway reaches the highest statistical significance. Bar color indicates statistical significance expressed as -log10(FDR). d AMPylation enrichment in H4-aSyn cells overexpressing FICD-WT (left) and FICD-E234G (right). Volcano plot depicts log2 fold change (pro-N6pA versus DMSO) versus –log10 p-value; dashed lines mark significance cut-offs (p < 0.01, -log2(pro-N6pA/DMSO) > 1). Significantly enriched AMPylated proteins are highlighted in blue. Representative lysosomal proteins and aSyn (SNCA) are annotated (for c - d: n = 2; n = 2 technical replicates / experiment). e Whole proteomic analysis and hierarchical clustering of proteome profiles of H4-Ctrl and H4-aSyn cells, either transfected with mock, FICD-WT, FICD-H363A, or FICD-E234G. Columns represent experimental conditions, partitioned into four clusters ( – ), while rows correspond to differentially regulated proteins (clusters I-IV). On the right, top 3 over-represented KEGG pathways for each row cluster are shown. Z-score: normalized expression scale from lower (blue) to higher (red) levels. The “protein processing in endoplasmic reticulum” pathway is overrepresented in row clusters I and II. f Heatmap showing expression of a subset of 23 proteins within the KEEG “protein processing in endoplasmic reticulum” pathway that are upregulated upon FICD-E234G overexpression (For subset identification, refer to Fig. S3i, cluster 5). Proteins involved in the UPR are highlighted in bold (for e - f: n = 3).

Figure Legend Snippet: a Experimental setup for investigating FICD and aSyn interaction in H4-Ctrl and H4-aSyn cells transiently transfected with FICD-WT, catalytically inactive FICD-H363A, or constitutively active FICD-E234G. Mock transfection served as baseline. b . Venn diagram of AMPylated proteins detected in H4-aSyn cells with mock, FICD-WT or FICD-E234G transfection. Overexpression of FICD-E234G resulted in an increase in AMPylated proteins. c KEGG pathway enrichment of AMPylated proteins in FICD-WT and FICD-E234G-transfected H4-aSyn cells. The “lysosome” pathway reaches the highest statistical significance. Bar color indicates statistical significance expressed as -log10(FDR). d AMPylation enrichment in H4-aSyn cells overexpressing FICD-WT (left) and FICD-E234G (right). Volcano plot depicts log2 fold change (pro-N6pA versus DMSO) versus –log10 p-value; dashed lines mark significance cut-offs (p < 0.01, -log2(pro-N6pA/DMSO) > 1). Significantly enriched AMPylated proteins are highlighted in blue. Representative lysosomal proteins and aSyn (SNCA) are annotated (for c - d: n = 2; n = 2 technical replicates / experiment). e Whole proteomic analysis and hierarchical clustering of proteome profiles of H4-Ctrl and H4-aSyn cells, either transfected with mock, FICD-WT, FICD-H363A, or FICD-E234G. Columns represent experimental conditions, partitioned into four clusters ( – ), while rows correspond to differentially regulated proteins (clusters I-IV). On the right, top 3 over-represented KEGG pathways for each row cluster are shown. Z-score: normalized expression scale from lower (blue) to higher (red) levels. The “protein processing in endoplasmic reticulum” pathway is overrepresented in row clusters I and II. f Heatmap showing expression of a subset of 23 proteins within the KEEG “protein processing in endoplasmic reticulum” pathway that are upregulated upon FICD-E234G overexpression (For subset identification, refer to Fig. S3i, cluster 5). Proteins involved in the UPR are highlighted in bold (for e - f: n = 3).

Techniques Used: Transfection, Over Expression, Expressing

a Schematic representation of the effect of FICD on BiP AMPylation with the downstream regulatory impact on UPR signaling branches IRE1α, PERK, and ATF6. b RT-qPCR analysis of canonical UPR markers in H4-Ctrl and H4-aSyn cells transfected with mock, FICD-WT, FICD-H363A, or FICD-E234G. mRNA expression of BiP, spliced XBP1 in the IRE1 branch, as well as PERK and CHOP in the PERK branch, is significantly upregulated upon FICD-E234G expression. Data are shown as expression relative to mock (dashed line) (n = 3 – 6). c CTSB activity assay normalized to mock (dashed line). Left, H4-Ctrl and H4-aSyn cells modulated by FICD variants. Right, FICD-modulated cells additionally exposed to cyclopiazonic acid (CPA; ER Ca 2+ -ATPase inhibitor) (n = 3). d Western blot showing CTSB pro-form (right top) and cleaved, mature CTSB heavy-chain (right bottom) in H4-Ctrl and H4-aSyn cells transfected with mock, FICD-WT, FICD-H363A, or FICD-E234G (n = 4). Bar graphs: mean ± SD. Statistical analysis: two-way ANOVA with Tukeýs multiple comparisons test. p* < 0.05, p** < 0.01, p*** < 0.001. LC: loading control via Coomassie brilliant blue; hc: heavy chain. sXBP1: spliced variant of XBP1; uXBP1: unspliced XBP1 .

Figure Legend Snippet: a Schematic representation of the effect of FICD on BiP AMPylation with the downstream regulatory impact on UPR signaling branches IRE1α, PERK, and ATF6. b RT-qPCR analysis of canonical UPR markers in H4-Ctrl and H4-aSyn cells transfected with mock, FICD-WT, FICD-H363A, or FICD-E234G. mRNA expression of BiP, spliced XBP1 in the IRE1 branch, as well as PERK and CHOP in the PERK branch, is significantly upregulated upon FICD-E234G expression. Data are shown as expression relative to mock (dashed line) (n = 3 – 6). c CTSB activity assay normalized to mock (dashed line). Left, H4-Ctrl and H4-aSyn cells modulated by FICD variants. Right, FICD-modulated cells additionally exposed to cyclopiazonic acid (CPA; ER Ca 2+ -ATPase inhibitor) (n = 3). d Western blot showing CTSB pro-form (right top) and cleaved, mature CTSB heavy-chain (right bottom) in H4-Ctrl and H4-aSyn cells transfected with mock, FICD-WT, FICD-H363A, or FICD-E234G (n = 4). Bar graphs: mean ± SD. Statistical analysis: two-way ANOVA with Tukeýs multiple comparisons test. p* < 0.05, p** < 0.01, p*** < 0.001. LC: loading control via Coomassie brilliant blue; hc: heavy chain. sXBP1: spliced variant of XBP1; uXBP1: unspliced XBP1 .

Techniques Used: Quantitative RT-PCR, Transfection, Expressing, Activity Assay, Western Blot, Control, Variant Assay

a Schematic overview of the SILAC pulse-chase experimental setup. H4-Ctrl and H4-aSyn cells were initially cultured in SILAC media containing light or heavy isotopes. Twenty-four hours after transfection with mock, FICD-WT, or FICD-E234G, the pulse-chase was initiated by switching the SILAC media to the opposite isotope condition (light-to-heavy or heavy-to-light). Isotope incorporation was monitored at multiple chase time points (2 h, 8 h, and 24 h) by LC-MS/MS to quantify the overall protein turnover. Depicted is the pulse-chase experimental workflow initiated with light SILAC medium. b Hierarchical clustering of half-lives from 938 commonly identified proteins. Z-score: normalized half-lives scale from lower (blue) to higher (red) levels. c Subcellular compartment analysis. Boxplots show distribution of protein half-lives grouped by GO cellular compartment annotation in H4-Ctrl (left) and H4-aSyn (right) cells. The x-axis shows the log2-transformed protein half-lives in log 2 (T1/2) [h]. Each box spans the interquartile range (IQR: 25 th -75 th percentile), with the center line indicating the median. Whiskers extend to data points within 1.5× IQR from the lower and upper quartiles, outliers are represented by dots. FICD-E234G overexpression significantly reduces global protein turnover compared with FICD-WT overexpression in both H4-Ctrl and H4-aSyn cells (n = 4). Statistical analysis: two-tailed Wilcoxon-matched-pairs signed rank test. p* < 0.05, p** < 0.01, p*** < 0.001, p**** < 0.0001.

Figure Legend Snippet: a Schematic overview of the SILAC pulse-chase experimental setup. H4-Ctrl and H4-aSyn cells were initially cultured in SILAC media containing light or heavy isotopes. Twenty-four hours after transfection with mock, FICD-WT, or FICD-E234G, the pulse-chase was initiated by switching the SILAC media to the opposite isotope condition (light-to-heavy or heavy-to-light). Isotope incorporation was monitored at multiple chase time points (2 h, 8 h, and 24 h) by LC-MS/MS to quantify the overall protein turnover. Depicted is the pulse-chase experimental workflow initiated with light SILAC medium. b Hierarchical clustering of half-lives from 938 commonly identified proteins. Z-score: normalized half-lives scale from lower (blue) to higher (red) levels. c Subcellular compartment analysis. Boxplots show distribution of protein half-lives grouped by GO cellular compartment annotation in H4-Ctrl (left) and H4-aSyn (right) cells. The x-axis shows the log2-transformed protein half-lives in log 2 (T1/2) [h]. Each box spans the interquartile range (IQR: 25 th -75 th percentile), with the center line indicating the median. Whiskers extend to data points within 1.5× IQR from the lower and upper quartiles, outliers are represented by dots. FICD-E234G overexpression significantly reduces global protein turnover compared with FICD-WT overexpression in both H4-Ctrl and H4-aSyn cells (n = 4). Statistical analysis: two-tailed Wilcoxon-matched-pairs signed rank test. p* < 0.05, p** < 0.01, p*** < 0.001, p**** < 0.0001.

Techniques Used: Multiplex sample analysis, Pulse Chase, Cell Culture, Transfection, Liquid Chromatography with Mass Spectroscopy, Transformation Assay, Over Expression, Two Tailed Test

$a Solubility assay of aSyn in H4-aSyn cells transfected with FICD-WT, -H363A, or -E234G using Western blot, analyzing aSyn in soluble (S) and insoluble (IS) fractions. Quantification shows elevated levels of insoluble aSyn upon FICD hyperactivation (n = 3). b Filter trap assay detecting aSyn aggregates using a pan-aSyn antibody (Syn1) and a conformation-dependent antibody with a higher affinity to aSyn aggregates (MJFR-14-6-4-2). Quantification demonstrates an increase in aSyn aggregation upon expression of FICD-E234G compared to FICD-WT or FICD-H363A (n = 6). Statistical analysis for a and b: one-way ANOVA with Tukeýs multiple comparisons test. c Flow cytometric analysis of apoptosis in H4-aSyn and H4-Ctrl cells. Cells were stained with Annexin V and propidium iodide (PI) to distinguish early (Q4) and late (Q2) apoptotic populations (highlighted in the left panel). Expression of FICD-E234G significantly increased early apoptosis in both cell lines, while late apoptosis was selectively elevated in H4-aSyn cells (n = 3). Statistical analysis: two-way ANOVA with Tukeýs multiple comparisons test. Bar graphs: mean ± SD. p* < 0.05, p** < 0.01, p*** < 0.001. Dashed lines: mock controls. LC: total protein via Coomassie brilliant blue for a) and Ponceau S for b); AV: Annexin V; PI: propidium iodide; IS: insoluble fraction; S: soluble fraction .$
Figure Legend Snippet: a Solubility assay of aSyn in H4-aSyn cells transfected with FICD-WT, -H363A, or -E234G using Western blot, analyzing aSyn in soluble (S) and insoluble (IS) fractions. Quantification shows elevated levels of insoluble aSyn upon FICD hyperactivation (n = 3). b Filter trap assay detecting aSyn aggregates using a pan-aSyn antibody (Syn1) and a conformation-dependent antibody with a higher affinity to aSyn aggregates (MJFR-14-6-4-2). Quantification demonstrates an increase in aSyn aggregation upon expression of FICD-E234G compared to FICD-WT or FICD-H363A (n = 6). Statistical analysis for a and b: one-way ANOVA with Tukeýs multiple comparisons test. c Flow cytometric analysis of apoptosis in H4-aSyn and H4-Ctrl cells. Cells were stained with Annexin V and propidium iodide (PI) to distinguish early (Q4) and late (Q2) apoptotic populations (highlighted in the left panel). Expression of FICD-E234G significantly increased early apoptosis in both cell lines, while late apoptosis was selectively elevated in H4-aSyn cells (n = 3). Statistical analysis: two-way ANOVA with Tukeýs multiple comparisons test. Bar graphs: mean ± SD. p* < 0.05, p** < 0.01, p*** < 0.001. Dashed lines: mock controls. LC: total protein via Coomassie brilliant blue for a) and Ponceau S for b); AV: Annexin V; PI: propidium iodide; IS: insoluble fraction; S: soluble fraction .

Techniques Used: Solubility, Transfection, Western Blot, TRAP Assay, Expressing, Staining

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Journal: bioRxiv

Article Title: Aberrant FICD-mediated AMPylation drives α-Synuclein pathology and overall protein dyshomeostasis in dopaminergic neurons in Parkinson’s disease

doi: 10.64898/2026.03.30.715195

Figure Lengend Snippet: a Experimental setup for investigating FICD and aSyn interaction in H4-Ctrl and H4-aSyn cells transiently transfected with FICD-WT, catalytically inactive FICD-H363A, or constitutively active FICD-E234G. Mock transfection served as baseline. b . Venn diagram of AMPylated proteins detected in H4-aSyn cells with mock, FICD-WT or FICD-E234G transfection. Overexpression of FICD-E234G resulted in an increase in AMPylated proteins. c KEGG pathway enrichment of AMPylated proteins in FICD-WT and FICD-E234G-transfected H4-aSyn cells. The “lysosome” pathway reaches the highest statistical significance. Bar color indicates statistical significance expressed as -log10(FDR). d AMPylation enrichment in H4-aSyn cells overexpressing FICD-WT (left) and FICD-E234G (right). Volcano plot depicts log2 fold change (pro-N6pA versus DMSO) versus –log10 p-value; dashed lines mark significance cut-offs (p < 0.01, -log2(pro-N6pA/DMSO) > 1). Significantly enriched AMPylated proteins are highlighted in blue. Representative lysosomal proteins and aSyn (SNCA) are annotated (for c - d: n = 2; n = 2 technical replicates / experiment). e Whole proteomic analysis and hierarchical clustering of proteome profiles of H4-Ctrl and H4-aSyn cells, either transfected with mock, FICD-WT, FICD-H363A, or FICD-E234G. Columns represent experimental conditions, partitioned into four clusters ( – ), while rows correspond to differentially regulated proteins (clusters I-IV). On the right, top 3 over-represented KEGG pathways for each row cluster are shown. Z-score: normalized expression scale from lower (blue) to higher (red) levels. The “protein processing in endoplasmic reticulum” pathway is overrepresented in row clusters I and II. f Heatmap showing expression of a subset of 23 proteins within the KEEG “protein processing in endoplasmic reticulum” pathway that are upregulated upon FICD-E234G overexpression (For subset identification, refer to Fig. S3i, cluster 5). Proteins involved in the UPR are highlighted in bold (for e - f: n = 3).

Article Snippet: H4 human neuroglioma cells (ATCC, HTB-148) stably expressing human wildtype (WT) aSyn (H4-aSyn) and control cells (H4-Ctrl) were generated using lentiviral vectors pCMV::aSyn-RES-GFP and pCMV::RES-GFP, respectively, as described previously ( ).

Techniques: Transfection, Over Expression, Expressing

Journal: bioRxiv

Article Title: Aberrant FICD-mediated AMPylation drives α-Synuclein pathology and overall protein dyshomeostasis in dopaminergic neurons in Parkinson’s disease

doi: 10.64898/2026.03.30.715195

Figure Lengend Snippet: a Schematic representation of the effect of FICD on BiP AMPylation with the downstream regulatory impact on UPR signaling branches IRE1α, PERK, and ATF6. b RT-qPCR analysis of canonical UPR markers in H4-Ctrl and H4-aSyn cells transfected with mock, FICD-WT, FICD-H363A, or FICD-E234G. mRNA expression of BiP, spliced XBP1 in the IRE1 branch, as well as PERK and CHOP in the PERK branch, is significantly upregulated upon FICD-E234G expression. Data are shown as expression relative to mock (dashed line) (n = 3 – 6). c CTSB activity assay normalized to mock (dashed line). Left, H4-Ctrl and H4-aSyn cells modulated by FICD variants. Right, FICD-modulated cells additionally exposed to cyclopiazonic acid (CPA; ER Ca 2+ -ATPase inhibitor) (n = 3). d Western blot showing CTSB pro-form (right top) and cleaved, mature CTSB heavy-chain (right bottom) in H4-Ctrl and H4-aSyn cells transfected with mock, FICD-WT, FICD-H363A, or FICD-E234G (n = 4). Bar graphs: mean ± SD. Statistical analysis: two-way ANOVA with Tukeýs multiple comparisons test. p* < 0.05, p** < 0.01, p*** < 0.001. LC: loading control via Coomassie brilliant blue; hc: heavy chain. sXBP1: spliced variant of XBP1; uXBP1: unspliced XBP1 .

Techniques: Quantitative RT-PCR, Transfection, Expressing, Activity Assay, Western Blot, Control, Variant Assay

Journal: bioRxiv

Article Title: Aberrant FICD-mediated AMPylation drives α-Synuclein pathology and overall protein dyshomeostasis in dopaminergic neurons in Parkinson’s disease

doi: 10.64898/2026.03.30.715195

Figure Lengend Snippet: a Schematic overview of the SILAC pulse-chase experimental setup. H4-Ctrl and H4-aSyn cells were initially cultured in SILAC media containing light or heavy isotopes. Twenty-four hours after transfection with mock, FICD-WT, or FICD-E234G, the pulse-chase was initiated by switching the SILAC media to the opposite isotope condition (light-to-heavy or heavy-to-light). Isotope incorporation was monitored at multiple chase time points (2 h, 8 h, and 24 h) by LC-MS/MS to quantify the overall protein turnover. Depicted is the pulse-chase experimental workflow initiated with light SILAC medium. b Hierarchical clustering of half-lives from 938 commonly identified proteins. Z-score: normalized half-lives scale from lower (blue) to higher (red) levels. c Subcellular compartment analysis. Boxplots show distribution of protein half-lives grouped by GO cellular compartment annotation in H4-Ctrl (left) and H4-aSyn (right) cells. The x-axis shows the log2-transformed protein half-lives in log 2 (T1/2) [h]. Each box spans the interquartile range (IQR: 25 th -75 th percentile), with the center line indicating the median. Whiskers extend to data points within 1.5× IQR from the lower and upper quartiles, outliers are represented by dots. FICD-E234G overexpression significantly reduces global protein turnover compared with FICD-WT overexpression in both H4-Ctrl and H4-aSyn cells (n = 4). Statistical analysis: two-tailed Wilcoxon-matched-pairs signed rank test. p* < 0.05, p** < 0.01, p*** < 0.001, p**** < 0.0001.

Techniques: Multiplex sample analysis, Pulse Chase, Cell Culture, Transfection, Liquid Chromatography with Mass Spectroscopy, Transformation Assay, Over Expression, Two Tailed Test

Journal: bioRxiv

Article Title: Aberrant FICD-mediated AMPylation drives α-Synuclein pathology and overall protein dyshomeostasis in dopaminergic neurons in Parkinson’s disease

doi: 10.64898/2026.03.30.715195

Figure Lengend Snippet: a Solubility assay of aSyn in H4-aSyn cells transfected with FICD-WT, -H363A, or -E234G using Western blot, analyzing aSyn in soluble (S) and insoluble (IS) fractions. Quantification shows elevated levels of insoluble aSyn upon FICD hyperactivation (n = 3). b Filter trap assay detecting aSyn aggregates using a pan-aSyn antibody (Syn1) and a conformation-dependent antibody with a higher affinity to aSyn aggregates (MJFR-14-6-4-2). Quantification demonstrates an increase in aSyn aggregation upon expression of FICD-E234G compared to FICD-WT or FICD-H363A (n = 6). Statistical analysis for a and b: one-way ANOVA with Tukeýs multiple comparisons test. c Flow cytometric analysis of apoptosis in H4-aSyn and H4-Ctrl cells. Cells were stained with Annexin V and propidium iodide (PI) to distinguish early (Q4) and late (Q2) apoptotic populations (highlighted in the left panel). Expression of FICD-E234G significantly increased early apoptosis in both cell lines, while late apoptosis was selectively elevated in H4-aSyn cells (n = 3). Statistical analysis: two-way ANOVA with Tukeýs multiple comparisons test. Bar graphs: mean ± SD. p* < 0.05, p** < 0.01, p*** < 0.001. Dashed lines: mock controls. LC: total protein via Coomassie brilliant blue for a) and Ponceau S for b); AV: Annexin V; PI: propidium iodide; IS: insoluble fraction; S: soluble fraction .

Techniques: Solubility, Transfection, Western Blot, TRAP Assay, Expressing, Staining

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