limp2 scarb2  (Cell Signaling Technology Inc)

Journal: Frontiers in Neurology

Article Title: Transcriptome and Proteome Analysis in LUHMES Cells Overexpressing Alpha-Synuclein

doi: 10.3389/fneur.2022.787059

Figure Lengend Snippet: Top 50 differentially regulated proteins between alpha-synuclein-overexpressing and GFP-expressing cells, sorted by Euclidian distance.

Article Snippet: For visualization of the proteins, the following primary antibodies were used: rabbit anti-ADD1 (1:500; Abcam, Cambridge United Kingdom), rabbit anti-ALDH6A1 (1:3,000; Proteintech, Rosemont, IL, USA), rabbit anti-ARHGEF2 (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit anti-ASNS (1:500; Proteintech), rabbit anti-αSyn (1:500; Thermo Fisher Scientific), goat anti-CNTN2 [1 μg/ml (R&D Systems, Minneapolis, MN, USA)], mouse anti-DLG4/PSD95 (1:500; Synaptic Systems, Göttingen, Germany), rabbit anti-EDF1 (1:500; Proteintech), mouse anti-LHX9 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), sheep anti-MDGA1 (0.1 μg/ml; R&D Systems), rabbit anti MTHFD2 (1:500; Proteintech), rabbit anti-NCALD (1:1,000; Proteintech), rabbit anti-PSAT1 (1:1,000; Proteintech), rabbit anti-SCARB2/LIMP2 (1:1,000; Novus Biologicals; Littleton, CO, USA), rabbit anti-SCG2 (1:1,000; Abcam), rabbit anti-SERPINB6 (1:500; Proteintech), rabbit anti-SHMT2 (1:1,000; Proteintech), rabbit anti-SLC7A1 (1:500; Proteintech), rabbit anti-STXBP1 (1:1,000; Synaptic Systems), mouse anti-TFRC (1:1,000; Thermo Fisher Scientific), rabbit anti-WARS1 (1:500; Thermo Fisher Scientific).

Techniques: Binding Assay, Membrane, Sequencing, RNA Binding Assay, Transduction

Journal: Frontiers in Neurology

Article Title: Transcriptome and Proteome Analysis in LUHMES Cells Overexpressing Alpha-Synuclein

doi: 10.3389/fneur.2022.787059

Figure Lengend Snippet: Regulation of the 21 transcripts/proteins that were differentially regulated between alpha-synuclein-overexpressing and GFP-expressing cells in the transcriptomic and proteomic level.

Article Snippet: For visualization of the proteins, the following primary antibodies were used: rabbit anti-ADD1 (1:500; Abcam, Cambridge United Kingdom), rabbit anti-ALDH6A1 (1:3,000; Proteintech, Rosemont, IL, USA), rabbit anti-ARHGEF2 (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit anti-ASNS (1:500; Proteintech), rabbit anti-αSyn (1:500; Thermo Fisher Scientific), goat anti-CNTN2 [1 μg/ml (R&D Systems, Minneapolis, MN, USA)], mouse anti-DLG4/PSD95 (1:500; Synaptic Systems, Göttingen, Germany), rabbit anti-EDF1 (1:500; Proteintech), mouse anti-LHX9 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), sheep anti-MDGA1 (0.1 μg/ml; R&D Systems), rabbit anti MTHFD2 (1:500; Proteintech), rabbit anti-NCALD (1:1,000; Proteintech), rabbit anti-PSAT1 (1:1,000; Proteintech), rabbit anti-SCARB2/LIMP2 (1:1,000; Novus Biologicals; Littleton, CO, USA), rabbit anti-SCG2 (1:1,000; Abcam), rabbit anti-SERPINB6 (1:500; Proteintech), rabbit anti-SHMT2 (1:1,000; Proteintech), rabbit anti-SLC7A1 (1:500; Proteintech), rabbit anti-STXBP1 (1:1,000; Synaptic Systems), mouse anti-TFRC (1:1,000; Thermo Fisher Scientific), rabbit anti-WARS1 (1:500; Thermo Fisher Scientific).

Techniques: Binding Assay

Journal: Frontiers in Neurology

Article Title: Transcriptome and Proteome Analysis in LUHMES Cells Overexpressing Alpha-Synuclein

doi: 10.3389/fneur.2022.787059

Figure Lengend Snippet: Western blots confirmation. Western blots with antibodies against the 21 proteins that were differentially regulated in the proteomics, of which the corresponding genes were also differentially regulated. For the Western blot investigation, samples from untransduced cells (untr), αSyn-overexpressing cells (αSyn), and GFP-expressing cells (GFP) cells were used. The proteins are presented in the same order as in . (A) SNCA, synuclein alpha; (B) SCG2, Secretogranin II; (C) MTHFD2, methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2, methenyltetrahydrofolate; (D) SHMT2, serine hydroxymethyltransferase 2; (E) WARS1, tryptophanyl-tRNA synthetase 1; (F) PSAT1, phosphoserine aminotransferase 1; (G) TFRC, transferrin receptor; (H) SERPINB6, serpin Family B, Member 6; (I) ASNS, asparagine synthetase (glutamine-hydrolyzing); (J) ARHGEF2, Rho/Rac guanine nucleotide exchange Factor 2; (K) SLC7A1, solute carrier Family 7, Member 1; (L) EDF1, endothelial differentiation-related Factor 1; (M) SCARB2, scavenger receptor Class B, Member 2; (N) CNTN2, contactin 2; (O) MDGA1, MAM domain containing glycosylphosphatidylinositol Anchor 1; (P) NCALD, neurocalcin delta; (Q) ADD1, Adducin 1; (R) STXBP1, syntaxin-binding Protein 1; (S) ALDH6A1, aldehyde dehydrogenase 6 Family, Member A1; (T) : LHX9, LIM Homeobox 9; (U) DLG4, discs large MAGUK scaffold Protein 4. Twenty out of 21 proteins (A–S,U) were differentially regulated between αSyn-overexpressing cells and GFP-expressing cells in the same direction as in the LC-MS investigation and thereby confirming these findings. With the exception of TFRC (G) , ADD1 (Q) , ALDH6A1 (S) , and DLG4 (U) , there was no difference in protein levels between untransduced cells and GFP-expressing cells, suggesting an αSyn-specific effect of the protein regulation. One protein (LHX9, T ) could not be confirmed. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. untransduced cells. § p < 0.05, §§ p < 0.01, §§§ p < 0.001 vs. GFP transduced cells.

Article Snippet: For visualization of the proteins, the following primary antibodies were used: rabbit anti-ADD1 (1:500; Abcam, Cambridge United Kingdom), rabbit anti-ALDH6A1 (1:3,000; Proteintech, Rosemont, IL, USA), rabbit anti-ARHGEF2 (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit anti-ASNS (1:500; Proteintech), rabbit anti-αSyn (1:500; Thermo Fisher Scientific), goat anti-CNTN2 [1 μg/ml (R&D Systems, Minneapolis, MN, USA)], mouse anti-DLG4/PSD95 (1:500; Synaptic Systems, Göttingen, Germany), rabbit anti-EDF1 (1:500; Proteintech), mouse anti-LHX9 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), sheep anti-MDGA1 (0.1 μg/ml; R&D Systems), rabbit anti MTHFD2 (1:500; Proteintech), rabbit anti-NCALD (1:1,000; Proteintech), rabbit anti-PSAT1 (1:1,000; Proteintech), rabbit anti-SCARB2/LIMP2 (1:1,000; Novus Biologicals; Littleton, CO, USA), rabbit anti-SCG2 (1:1,000; Abcam), rabbit anti-SERPINB6 (1:500; Proteintech), rabbit anti-SHMT2 (1:1,000; Proteintech), rabbit anti-SLC7A1 (1:500; Proteintech), rabbit anti-STXBP1 (1:1,000; Synaptic Systems), mouse anti-TFRC (1:1,000; Thermo Fisher Scientific), rabbit anti-WARS1 (1:500; Thermo Fisher Scientific).

Techniques: Western Blot, Expressing, Binding Assay, Liquid Chromatography with Mass Spectroscopy

Journal: Frontiers in Neurology

Article Title: Transcriptome and Proteome Analysis in LUHMES Cells Overexpressing Alpha-Synuclein

doi: 10.3389/fneur.2022.787059

Figure Lengend Snippet: Overlap to existing datasets. (A) Overlaps between N = 357 genes that were associated with risk loci in two recent Parkinson's disease genome-wide associated studies (GWAS) (an orange circle) and transcripts (a yellow circle) and protein (a blue circle) that were differentially regulated in our cell model. Of the 357 genes associated with PD, N = 11 were differentially regulated in our cell model (a brown area). Furthermore, N = 7 proteins encoded by these genes were differentially regulated in our cell model (a pink area). In addition to SNCA (alpha-synuclein) that was overexpressed in our cells model, also SCARB2 (scavenger receptor Class B, Member 2) was regulated on the transcriptomic and proteomic level (a red area). (B) Overlap between N = 434 genes present in the human lysosome gene database (an orange circle) and differentially regulated genes (a yellow circle) or proteins (a blue circle) in our cell model. N = 15 of the 434 lysosomal genes were differentially regulated in our cell model (a brown area). N = 11 proteins that were present in the human lysosome gene database were differentially regulated in our cell model (a pink area). Red characters indicate genes/proteins (SCARB2, scavenger receptor class B, member 2; CTSB, cathepsin B) that were found in the recent GWAS meta-analyses. (C) Overlap between N = 1,233 synaptic genes (an orange circle) and genes (a yellow circle) or proteins (a blue circle) that were regulated in our cell model. N = 61 synaptic genes were differentially regulated in our cell model (a brown area). Furthermore, N = 25 of the proteins that were differentially regulated in our cell model were encoded by synaptic genes (a pink area). Red characters mark genes/proteins that have been found in the recent GWAS meta-analyses (FYN, FYN proto-oncogene, Src family tyrosine kinase; STX1B, syntaxin 1B). In addition to SNCA, 7 more genes/proteins were regulated on the transcriptomic and proteomic level in our cell model (a red area). Diff. reg., differentially regulated (adjusted p -value < 0.05, Benjanimi-Hochberg false discovery rate).

Article Snippet: For visualization of the proteins, the following primary antibodies were used: rabbit anti-ADD1 (1:500; Abcam, Cambridge United Kingdom), rabbit anti-ALDH6A1 (1:3,000; Proteintech, Rosemont, IL, USA), rabbit anti-ARHGEF2 (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit anti-ASNS (1:500; Proteintech), rabbit anti-αSyn (1:500; Thermo Fisher Scientific), goat anti-CNTN2 [1 μg/ml (R&D Systems, Minneapolis, MN, USA)], mouse anti-DLG4/PSD95 (1:500; Synaptic Systems, Göttingen, Germany), rabbit anti-EDF1 (1:500; Proteintech), mouse anti-LHX9 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), sheep anti-MDGA1 (0.1 μg/ml; R&D Systems), rabbit anti MTHFD2 (1:500; Proteintech), rabbit anti-NCALD (1:1,000; Proteintech), rabbit anti-PSAT1 (1:1,000; Proteintech), rabbit anti-SCARB2/LIMP2 (1:1,000; Novus Biologicals; Littleton, CO, USA), rabbit anti-SCG2 (1:1,000; Abcam), rabbit anti-SERPINB6 (1:500; Proteintech), rabbit anti-SHMT2 (1:1,000; Proteintech), rabbit anti-SLC7A1 (1:500; Proteintech), rabbit anti-STXBP1 (1:1,000; Synaptic Systems), mouse anti-TFRC (1:1,000; Thermo Fisher Scientific), rabbit anti-WARS1 (1:500; Thermo Fisher Scientific).

Techniques: Genome Wide