Journal: Journal of proteome research

Article Title: Quantitative Proteomics Reveal an Altered Pattern of Protein Expression in Brain Tissue from Mice Lacking GPR37 and GPR37L1

doi: 10.1021/acs.jproteome.9b00622

Figure Lengend Snippet: (A) Co-expression of S100A5 with either GPR37 or GPR37L1 in HEK293T cells did not significantly increase intracellular S100A5 expression (n=12 per condition, bars represent SEM, one-way ANOVA). (B) Co-expression of S100A5 with either GPR37 or GPR37L1 did increase S100A5 secretion compared to S100A5 expression alone (n=7, bars represent SEM one-way ANOVA, ****: p<0.0001). (C) Representative Western blot of cell lysates and media is shown on the right demonstrating expression of transfected proteins (cell lysate) and secretion (media) of S100A5 into the media. HEK293T cells were transfected to co-express S100A5 in the absence and presence of GRP37 or GPR37L1.

Article Snippet: Constructs used included the following: empty vector, human GPR37L1 (untagged), human GPR37 (untagged), human S100A5-myc-DDK (Origene), human S100A4-myc-DDK (Origene), and human S100A6-myc-DDK (Origene).

Techniques: Expressing, Western Blot, Transfection

Journal: Journal of proteome research

Article Title: Quantitative Proteomics Reveal an Altered Pattern of Protein Expression in Brain Tissue from Mice Lacking GPR37 and GPR37L1

doi: 10.1021/acs.jproteome.9b00622

Figure Lengend Snippet: (A) To assess whether secretion of S100A5 can be induced by simply overexpressing the protein, increasing amounts of S100A5 plasmid (0.5 μg up to 6μg) were transfected into HEK293T cells. However, increasing S100A5 levels in HEK293T cells did not lead to detectable S100A5 secretion, suggesting that secretion of S100A5 is receptor-dependent. (B) Secretion of S100A5 increased according to the amount of receptor co-expressed (this panel shows a quantification of the data shown in panels C and D). (C) Representative Western blot depicting the effect of increasing GPR37L1 levels on S100A5 secretion (n=4). (D) Representative Western blot depicting effect of increasing GPR37 levels on S100A5 secretion (n=4). All samples were normalized to GAPDH. Bars represent SEM.

Article Snippet: Constructs used included the following: empty vector, human GPR37L1 (untagged), human GPR37 (untagged), human S100A5-myc-DDK (Origene), human S100A4-myc-DDK (Origene), and human S100A6-myc-DDK (Origene).

Techniques: Plasmid Preparation, Transfection, Western Blot

Journal: Journal of proteome research

Article Title: Quantitative Proteomics Reveal an Altered Pattern of Protein Expression in Brain Tissue from Mice Lacking GPR37 and GPR37L1

doi: 10.1021/acs.jproteome.9b00622

Figure Lengend Snippet: To elucidate the mechanism regulating S100A5 secretion, HEK293T cells were transfected with S100A5 and either GPR37 or GPR37L1. Cells were allowed to incubate for 24 hours following transfection and then treated with different pharmacological reagents. (A) Normalized quantification of S100A5 secretion when cells were treated with either BAPTA-AM or the putative GPR37L1 ligand prosaptide (TX 14A) compared to transfected cells that did not receive treatment. (B) Representative Western blots depicting effects of increasing concentrations of BAPTA-AM on S100A5 secretion in cells co-expressing S100A5 and GPR37L1. (C) Representative Western blot depicting effects of increasing concentrations of BAPTA-AM on S100A5 secretion in cells co-expressing S100A5 and GPR37. (n=3-4, bars represent SEM, two-way ANOVA within groups, Dunnett’s post hoc) (*p=0.0110; **p<0.006; ****p<0.0001)

Article Snippet: Constructs used included the following: empty vector, human GPR37L1 (untagged), human GPR37 (untagged), human S100A5-myc-DDK (Origene), human S100A4-myc-DDK (Origene), and human S100A6-myc-DDK (Origene).

Techniques: Transfection, Western Blot, Expressing

Journal: Journal of proteome research

Article Title: Quantitative Proteomics Reveal an Altered Pattern of Protein Expression in Brain Tissue from Mice Lacking GPR37 and GPR37L1

doi: 10.1021/acs.jproteome.9b00622

Figure Lengend Snippet: To explore whether GPR37- or GPR37L1-mediated secretion was specific only to S100A5 or general for other S100A protein family members, we examined whether co-expression of either GPR37 or GPR37L1 with S100A4 or S100A10 in HEK293T cells might lead to secretion of these proteins. (A) S100A4 co-expression with either receptor resulted in detectable secretion. (B) In contrast, S100A10 secretion was not observed upon co-expression with either receptor. The positive control shown here was a media sample containing secreted S100A5.

Article Snippet: Constructs used included the following: empty vector, human GPR37L1 (untagged), human GPR37 (untagged), human S100A5-myc-DDK (Origene), human S100A4-myc-DDK (Origene), and human S100A6-myc-DDK (Origene).

Techniques: Expressing, Positive Control

Journal: Journal of proteome research

Article Title: Quantitative Proteomics Reveal an Altered Pattern of Protein Expression in Brain Tissue from Mice Lacking GPR37 and GPR37L1

doi: 10.1021/acs.jproteome.9b00622

Figure Lengend Snippet: (A) Volcano plot depicting all protein hits from proteomic analyses comparing global protein changes between DKO and WT mouse brains. Whole mouse brain lysates were processed at the Emory Proteomics Core for analysis via mass spectrometry. The x-axis represents fold change (increased, positive Log2(DKO/WT) or decreased, negative Log2(DKO/WT)) and the y-axis represents −log10(p value), i.e. the bigger the −log10(p value), the more significant the data point is. The proteomic screen yielded only a small handful of proteins that exhibited both a significant and high fold change. Data points colored in red are those proteins that were significantly decreased (p≤0.05, DKO-WT Log2 ≤ −0.36) in DKO mouse brains compared to WT mouse brains, and data points colored in green are those that were significantly increased (p≤0.05, DKO-WT Log2 ≥ 0.36). Out of over 4,500 proteomic hits, only 78 were significantly increased (greater than or equal to +28.6% fold change) and only 53 were significantly decreased (less than or equal to −28.6% fold change) when both GPR37 and GPR37L1 were knocked out. WT, n=3; DKO, n=3. (B) A protein-protein interaction network between the 78 increased and 53 decreased proteins was generated via STRING analysis (string-db.org). This network identifies any protein-protein interactions determined experimentally (blue lines) or found in curated databases (gray lines). (C,D) Ontology graphs were generated each for the increased (C) and increased and decreased combined (D) lists of proteins.

Article Snippet: Constructs used included the following: empty vector, human GPR37L1 (untagged), human GPR37 (untagged), human S100A5-myc-DDK (Origene), human S100A4-myc-DDK (Origene), and human S100A6-myc-DDK (Origene).

Techniques: Mass Spectrometry, Generated

Journal: Journal of proteome research

Article Title: Quantitative Proteomics Reveal an Altered Pattern of Protein Expression in Brain Tissue from Mice Lacking GPR37 and GPR37L1

doi: 10.1021/acs.jproteome.9b00622

Figure Lengend Snippet: Top 7 most significantly decreased proteins in GPR37/GPR37L1 double knockout (DKO) vs. wild-type (WT) whole mouse brain identified via mass spectrometry

Article Snippet: Constructs used included the following: empty vector, human GPR37L1 (untagged), human GPR37 (untagged), human S100A5-myc-DDK (Origene), human S100A4-myc-DDK (Origene), and human S100A6-myc-DDK (Origene).

Techniques: Double Knockout

Journal: Journal of proteome research

Article Title: Quantitative Proteomics Reveal an Altered Pattern of Protein Expression in Brain Tissue from Mice Lacking GPR37 and GPR37L1

doi: 10.1021/acs.jproteome.9b00622

Figure Lengend Snippet: Top 9 most significantly increased proteins in GPR37/GPR37L1 double knockout (DKO) vs. wild-type (WT) whole mouse brain identified via mass spectrometry

Article Snippet: Constructs used included the following: empty vector, human GPR37L1 (untagged), human GPR37 (untagged), human S100A5-myc-DDK (Origene), human S100A4-myc-DDK (Origene), and human S100A6-myc-DDK (Origene).

Techniques: Double Knockout