Journal: Frontiers in Cellular Neuroscience

Article Title: Proteomic and Metabolomic Analyses of Vanishing White Matter Mouse Astrocytes Reveal Deregulation of ER Functions

doi: 10.3389/fncel.2017.00411

Figure Lengend Snippet: Primers used for qPCR.

Article Snippet: Membranes were blocked in 5% (w/v) milk powder and stained with the antibody against PROS1 (16910-1-AP, Proteintech) or SLC3A2 (LS-C334231, LSBio) overnight (16 h) at 4°C.

Techniques:

Journal: Frontiers in Cellular Neuroscience

Article Title: Proteomic and Metabolomic Analyses of Vanishing White Matter Mouse Astrocytes Reveal Deregulation of ER Functions

doi: 10.3389/fncel.2017.00411

Figure Lengend Snippet: Primer sequences used for cloning.

Article Snippet: Membranes were blocked in 5% (w/v) milk powder and stained with the antibody against PROS1 (16910-1-AP, Proteintech) or SLC3A2 (LS-C334231, LSBio) overnight (16 h) at 4°C.

Techniques: Cloning, Construct, Sequencing

Journal: Frontiers in Cellular Neuroscience

Article Title: Proteomic and Metabolomic Analyses of Vanishing White Matter Mouse Astrocytes Reveal Deregulation of ER Functions

doi: 10.3389/fncel.2017.00411

Figure Lengend Snippet: Volcano plot of all 1240 proteins from the proteomic screen and DAVID analysis of the 80 proteins regulated by eIF2B𝜀 Arg191His identifies a difference in proteins that migrate through the secretory pathway. (A) In the volcano plot the proteins in darker gray have a p -value < 0.05 and are considered significantly differently synthesized between wt and 2b5 ho astrocyte cultures. The p -values are plotted as Log(10) and p < 0.05 corresponds to -Log10 > 1.12. The fold change between wt and 2b5 ho is plotted as Log(2) and a fold change of >2 corresponds to Log(2) > 1. 80 proteins are significantly different between wt and 2b5 ho astrocytes of which 72 are increased and 8 are decreased in 2b5 ho astrocytes. The proteins SLC3A2, PROS1, and GAS6 are highlighted in black. (B) The figure shows the overrepresentation analysis ( p -value < 0.05) between the significantly different proteins with the proteins found in the proteome as a background. The pathway analysis reveals an overrepresentation of proteins with a signal peptide, transmembrane domain, N -linked glycosylation site(s) and/or disulfide bond(s). These terms together pinpoint targeting of proteins to the secretory pathway, some of which are post-translationally modified, as being affected by this mutation.

Article Snippet: Membranes were blocked in 5% (w/v) milk powder and stained with the antibody against PROS1 (16910-1-AP, Proteintech) or SLC3A2 (LS-C334231, LSBio) overnight (16 h) at 4°C.

Techniques: Synthesized, Glycoproteomics, Modification, Mutagenesis

Journal: Frontiers in Cellular Neuroscience

Article Title: Proteomic and Metabolomic Analyses of Vanishing White Matter Mouse Astrocytes Reveal Deregulation of ER Functions

doi: 10.3389/fncel.2017.00411

Figure Lengend Snippet: mRNA and protein levels of candidate proteins in astrocyte cultures. (A) mRNA levels of the investigated candidate were investigated with qPCR. The mRNA levels were similar between wt and 2b5 ho astrocytes ( n ≥ 5), with the exception of the Fndc3c1 mRNA, which was increased in 2b5 ho astrocytes. (B) The total protein levels of PROS1 and SLC3A2 were determined by Western blot. The graph shows the mean ± SD. The protein levels were increased in 2b5 ho cultures compared to wt cultures ( n ≥ 3). ∗∗ p < 0.01. Post-translational modifications were overall not affected in 2b5 ho astrocytes. Western blot analyses did not show whether PROS1 in astrocytes was post-translationally modified or not. Staining of all proteins (loading control) is shown in Supplementary Figure .

Article Snippet: Membranes were blocked in 5% (w/v) milk powder and stained with the antibody against PROS1 (16910-1-AP, Proteintech) or SLC3A2 (LS-C334231, LSBio) overnight (16 h) at 4°C.

Techniques: Western Blot, Modification, Staining, Control

Journal: Frontiers in Cellular Neuroscience

Article Title: Proteomic and Metabolomic Analyses of Vanishing White Matter Mouse Astrocytes Reveal Deregulation of ER Functions

doi: 10.3389/fncel.2017.00411

Figure Lengend Snippet: In vitro analyses of candidate gene expression regulation do not reveal difference in promoter or 5′-UTR efficiency in wt and 2b5 ho astrocytes. pNL1.1- Scrn1 , - Gas6 and - Pros1 constructs and pNL1.1- Sorcs1 , - Ddr1 , - Gas6 and - Pros1 5′UTR constructs were transfected into wt and 2b5 ho astrocytes. Nanoluciferase activity was measured 3 days post transfection and corrected for firefly luciferase activity expressed from the pGL3- Gapdh internal standard. Every culture is shown as a single dot and bars represent the mean ± SD ( n = 5–9). Replacement of the authentic Pros1 or Gas6 promoter by the Gapdh promoter increased the nanoluciferase expression by approximately twofold. pNL1.1- Gapdh was used as reference non-regulated promoter.

Article Snippet: Membranes were blocked in 5% (w/v) milk powder and stained with the antibody against PROS1 (16910-1-AP, Proteintech) or SLC3A2 (LS-C334231, LSBio) overnight (16 h) at 4°C.

Techniques: In Vitro, Gene Expression, Construct, Transfection, Activity Assay, Luciferase, Expressing