Journal: Biochemistry

Article Title: N-Linked Glycosylation-Dependent and -Independent Mechanisms Regulating CTRP12 Cleavage, Secretion, and Stability

doi: 10.1021/acs.biochem.8b00528

Figure Lengend Snippet: Inhibition of N-glycosylation enhances CTRP12 cleavage. (A) Domain structure of mouse CTRP12. The conserved furin cleavage site (Lys-91) and the three potential N-linked glycosylation sites (Asn-39, Asn-287, and Asn-297) that conform to the N-X-S/T motif are indicated. The C-terminal FLAG tag is also indicated. (B) Western blot analysis (using anti-FLAG M2 antibody) of the cell lysate and supernatant of transfected HEK293 cells treated with vehicle control or tunicamycin, an inhibitor of N-glycosylation. (C) Quantification of the ratio of cleaved to full-length protein in the supernatant. FL, full-length; N = 6 (each lane represents a different sample from an independent transfection). ****p < 0.0001.

Article Snippet: All C-terminal FLAG epitope-tagged mouse CTRP12 (NCBI GenBank entry {"type":"entrez-protein","attrs":{"text":"NP_080401","term_id":"13385632","term_text":"NP_080401"}} NP_080401 ) single-, double-, and triple-mutant constructs (N39Q, N39A, N287Q, N287A, N297Q, N297A, T41A, S289A, S299A, N39Q/N287Q, N39Q/N297Q, N287Q/N297Q, and N39Q/N287Q/N297Q) were generated by either site-directed mutagenesis or GenScript.

Techniques: Inhibition, FLAG-tag, Western Blot, Transfection

Journal: Biochemistry

Article Title: N-Linked Glycosylation-Dependent and -Independent Mechanisms Regulating CTRP12 Cleavage, Secretion, and Stability

doi: 10.1021/acs.biochem.8b00528

Figure Lengend Snippet: Mass spectrometry and mutational analysis of N-glycosylation in CTRP12. (A) MS/MS spectrum of the glycopeptide ARVDSPNITTSNR derived from CTRP12 identified using Proteome Discoverer. Higher-energy collisional dissociation (HCD) generated b- (red) and y- (blue) peptide fragments that allow for the mapping of the site of glycosylation as indicated by the asterisk. Cleavage of the glycan structure from the peptide backbone via PNGase F digestion results in deamidation of asparagine and conversion to aspartic acid. Mass spectra indicate that Asn-39 is N-glycosylated. (B) Western blot analysis of cell lysates of transfected HEK293 cells expressing C-terminal FLAG-tagged wild-type (WT) CTRP12 or a single mutant (N39Q, N297Q, or N287Q). (C) Western blot analysis of cell lysates of transfected HEK293 cells expressing N-terminal FLAG-tagged WT CTRP12 or a single mutant (N297Q or N287Q).

Article Snippet: All C-terminal FLAG epitope-tagged mouse CTRP12 (NCBI GenBank entry {"type":"entrez-protein","attrs":{"text":"NP_080401","term_id":"13385632","term_text":"NP_080401"}} NP_080401 ) single-, double-, and triple-mutant constructs (N39Q, N39A, N287Q, N287A, N297Q, N297A, T41A, S289A, S299A, N39Q/N287Q, N39Q/N297Q, N287Q/N297Q, and N39Q/N287Q/N297Q) were generated by either site-directed mutagenesis or GenScript.

Techniques: Mass Spectrometry, Tandem Mass Spectroscopy, Derivative Assay, Generated, Western Blot, Transfection, Expressing, Mutagenesis

Journal: Biochemistry

Article Title: N-Linked Glycosylation-Dependent and -Independent Mechanisms Regulating CTRP12 Cleavage, Secretion, and Stability

doi: 10.1021/acs.biochem.8b00528

Figure Lengend Snippet: Asn-39 glycosylation regulates CTRP12 cleavage and stability. Western blot analysis of the cell lysate and supernatant of transfected HEK293 cells expressing wild-type (WT) CTRP12 or the (A) N39Q, (D) N39A, and (G) T41A mutants. Quantification of the ratio of cleaved to full-length (FL) protein (the cleavage index) in the supernatant for (B) N39Q, (E) N39A, and (H) T41A mutants. Quantification of the ratio of secreted to intracellular CTRP12 (the secretion index) for (C) N39Q, (F) 39A, and (I) T41 mutants. (J) A 6 h cycloheximide chase analysis of WT and the N39Q mutant. (K) Quantification of the relative protein stability of N39Q relative to WT CTRP12. (L) A 6 h cycloheximide chase analysis of the T41A mutant. (M) Quantification of the relative protein stability of T41A relative to WT CTRP12. N = 6 (each lane represents a different sample from an independent transfection). **p < 0.01. ***p < 0.001. ****p < 0.0001.

Article Snippet: All C-terminal FLAG epitope-tagged mouse CTRP12 (NCBI GenBank entry {"type":"entrez-protein","attrs":{"text":"NP_080401","term_id":"13385632","term_text":"NP_080401"}} NP_080401 ) single-, double-, and triple-mutant constructs (N39Q, N39A, N287Q, N287A, N297Q, N297A, T41A, S289A, S299A, N39Q/N287Q, N39Q/N297Q, N287Q/N297Q, and N39Q/N287Q/N297Q) were generated by either site-directed mutagenesis or GenScript.

Techniques: Western Blot, Transfection, Expressing, Mutagenesis

Journal: Biochemistry

Article Title: N-Linked Glycosylation-Dependent and -Independent Mechanisms Regulating CTRP12 Cleavage, Secretion, and Stability

doi: 10.1021/acs.biochem.8b00528

Figure Lengend Snippet: Nonglycosylated Asn-297 independently regulates CTRP12 cleavage and stability. Western blot analysis of the cell lysate and supernatant of transfected HEK293 cells expressing wild-type (WT) CTRP12 or the (A) N297Q, (D) N297A, and (G) S299A mutants. Quantification of the ratio of cleaved to full-length (FL) protein (the cleavage index) in the supernatant for (B) N297Q and (E) N297A. Quantification of the ratio of secreted to intracellular CTRP12 protein (the secretion index) for (C) N297Q and (F) N297A. (H) A 6 h cycloheximide chase analysis of the N297Q mutant. (I) Quantification of the relative protein stability of N297Q relative to WT CTRP12 (WT data shown in Figure 3J). N = 6 (each lane represents a different sample from an independent transfection). *p < 0.05. ***p < 0.001. ****p < 0.0001.

Article Snippet: All C-terminal FLAG epitope-tagged mouse CTRP12 (NCBI GenBank entry {"type":"entrez-protein","attrs":{"text":"NP_080401","term_id":"13385632","term_text":"NP_080401"}} NP_080401 ) single-, double-, and triple-mutant constructs (N39Q, N39A, N287Q, N287A, N297Q, N297A, T41A, S289A, S299A, N39Q/N287Q, N39Q/N297Q, N287Q/N297Q, and N39Q/N287Q/N297Q) were generated by either site-directed mutagenesis or GenScript.

Techniques: Western Blot, Transfection, Expressing, Mutagenesis

Journal: Biochemistry

Article Title: N-Linked Glycosylation-Dependent and -Independent Mechanisms Regulating CTRP12 Cleavage, Secretion, and Stability

doi: 10.1021/acs.biochem.8b00528

Figure Lengend Snippet: Impact of the hexosamine biosynthesis pathway on CTRP12 cleavage and stability. (A and B) Quantification of glycosylated proteins (relative to total) in HEK293 cells treated with 5 mM glucosamine (GlcN) for 48 h using Pro-Q emerald 300 and biotin-conjugated concanavalin A, respectively. Each lane represents an independent biological sample. (C) Western blot quantification of three major ER stress pathways (XBP1, CHOP/GADD153, and ATF-6) in HEK293 cells treated with the vehicle control or 5 mM glucosamine. XBP-1s refers to the spliced isoform of XBP-1. (D) Western blot analysis of the cell lysate and supernatant of transfected HEK293 cells expressing wild-type (WT) CTRP12 treated with 5 mM glucosamine. (E) Quantification of the ratio of cleaved to full-length (FL) protein (the cleavage index) in the supernatant of cells treated with glucosamine. (F) Quantification of the ratio of secreted to intracellular CTRP12 protein (the secretion index) as shown in panel D. (G) A 6 h cycloheximide chase experiment with HEK293 cells treated with 5 mM glucosamine. (H) Quantification of the relative protein stability of CTRP12 in glucosamine-treated cells vs nontreated controls (same as the top panel in Figure 3J). N = 6 (each lane represents a different sample from an independent transfection). **p < 0.01. ***p < 0.001. ****p < 0.0001.

Article Snippet: All C-terminal FLAG epitope-tagged mouse CTRP12 (NCBI GenBank entry {"type":"entrez-protein","attrs":{"text":"NP_080401","term_id":"13385632","term_text":"NP_080401"}} NP_080401 ) single-, double-, and triple-mutant constructs (N39Q, N39A, N287Q, N287A, N297Q, N297A, T41A, S289A, S299A, N39Q/N287Q, N39Q/N297Q, N287Q/N297Q, and N39Q/N287Q/N297Q) were generated by either site-directed mutagenesis or GenScript.

Techniques: Western Blot, Transfection, Expressing

Journal: Biochemistry

Article Title: N-Linked Glycosylation-Dependent and -Independent Mechanisms Regulating CTRP12 Cleavage, Secretion, and Stability

doi: 10.1021/acs.biochem.8b00528

Figure Lengend Snippet: Complex-type glycans influence CTRP12 cleavage. (A) PCR amplification of human GnTI/MGAT1 mRNA in wild-type HEK293 cells and GnTI-deficient HEK293S cells. The ~1.5 kb amplicon includes the entire coding region of GnTI. (B) Western blot analysis of endogenous GnTI protein in wild-type (WT) HEK293 cells and GnTI-deficient HEK293S cells. (C and D) Immunoblot analysis (using anti-FLAG antibody) of CTRP12 expressed in HEK293 (GnTI +) and HEK293S (GnTI −) cells. Cell lysates were subjected to PNGaseF (C) or endoglycosidase H (D) treatment before Western blot analysis. (E) Western blot analysis of the cell lysate and supernatant of transfected HEK293 cells with (+) or without (−) N-acetylglucosaminyltransferase I (GnTI) activity involved in N-glycan remodeling in the Golgi compartment. (F) Quantification of the ratio of cleaved to full-length protein (the cleavage index) in the supernatant. (G) Quantification of the ratio of secreted to intracellular CTRP12 (the secretion index). (H) Western blot analysis of the cell lysate and supernatant of GnTI-deficient HEK293 cells re-expressing WT human GnTI/MGAT1. (I) Quantification of the ratio of cleaved to full-length protein (the cleavage index) in the supernatant. (J) Quantification of the ratio of secreted to intracellular CTRP12 (the secretion index). N = 6 (each lane represents a different sample from an independent transfection). *p < 0.05. ****p < 0.0001.

Article Snippet: All C-terminal FLAG epitope-tagged mouse CTRP12 (NCBI GenBank entry {"type":"entrez-protein","attrs":{"text":"NP_080401","term_id":"13385632","term_text":"NP_080401"}} NP_080401 ) single-, double-, and triple-mutant constructs (N39Q, N39A, N287Q, N287A, N297Q, N297A, T41A, S289A, S299A, N39Q/N287Q, N39Q/N297Q, N287Q/N297Q, and N39Q/N287Q/N297Q) were generated by either site-directed mutagenesis or GenScript.

Techniques: Amplification, Western Blot, Transfection, Activity Assay, Expressing

Journal: Biochemistry

Article Title: N-Linked Glycosylation-Dependent and -Independent Mechanisms Regulating CTRP12 Cleavage, Secretion, and Stability

doi: 10.1021/acs.biochem.8b00528

Figure Lengend Snippet: Nonglycosylated Asn-287 is required for proper protein folding and secretion. (A and B) Western blot analysis of the cell lysate and supernatant of transfected HEK293 cells expressing wild-type (WT) CTRP12 and the N287Q and N287A mutants, respectively. (C) Western blot analysis of Endo H-digested CTRP12 (N287Q mutant) found in the transfected cell lysate. (D) Formation of the higher-molecular weight protein aggregate of the N287Q mutant in transfected cells. Cell lysates were heated at 95 °C for 5 min in the presence (+) or absence (−) of reducing agent β-ME (β-mercaptoethanol) prior to Western blotting. (E) Western blot analysis of the cell lysate and supernatant of transfected HEK293 cells expressing the S289A mutant. N = 6 (each lane represents a different sample from an independent transfection).

Article Snippet: All C-terminal FLAG epitope-tagged mouse CTRP12 (NCBI GenBank entry {"type":"entrez-protein","attrs":{"text":"NP_080401","term_id":"13385632","term_text":"NP_080401"}} NP_080401 ) single-, double-, and triple-mutant constructs (N39Q, N39A, N287Q, N287A, N297Q, N297A, T41A, S289A, S299A, N39Q/N287Q, N39Q/N297Q, N287Q/N297Q, and N39Q/N287Q/N297Q) were generated by either site-directed mutagenesis or GenScript.

Techniques: Western Blot, Transfection, Expressing, Mutagenesis, Molecular Weight

Journal: Biochemistry

Article Title: N-Linked Glycosylation-Dependent and -Independent Mechanisms Regulating CTRP12 Cleavage, Secretion, and Stability

doi: 10.1021/acs.biochem.8b00528

Figure Lengend Snippet: Furin-mediated cleavage of CTRP12. (A) Western blot analysis of the cell lysate and supernatant of transfected HEK293 cells expressing the maximally cleaved CTRP12 double mutant (N39Q/N297Q) treated with the vehicle control or furin/PCSK3 inhibitor. (B) Quantification of the ratio of cleaved to full-length (FL) protein (the cleavage index) in the supernatant as shown in panel A. (C) Western blot analysis of the cell lysate and supernatant of transfected LoVo cells co-expressing the maximally cleaved CTRP12 double mutant (N39Q/N297Q) and mouse furin. The LoVo cell lacks a functional furin gene, and hence, the double mutant is not cleaved. Furin re-expression restores maximal cleavage. (D) Quantification of the ratio of cleaved to full-length (FL) protein (the cleavage index) in the supernatant as shown in panel C. N = 6 (each lane represents a different sample from an independent transfection). **p < 0.01. ****p < 0.0001.

Article Snippet: All C-terminal FLAG epitope-tagged mouse CTRP12 (NCBI GenBank entry {"type":"entrez-protein","attrs":{"text":"NP_080401","term_id":"13385632","term_text":"NP_080401"}} NP_080401 ) single-, double-, and triple-mutant constructs (N39Q, N39A, N287Q, N287A, N297Q, N297A, T41A, S289A, S299A, N39Q/N287Q, N39Q/N297Q, N287Q/N297Q, and N39Q/N287Q/N297Q) were generated by either site-directed mutagenesis or GenScript.

Techniques: Western Blot, Transfection, Expressing, Mutagenesis, Functional Assay

Journal: Biochemistry

Article Title: N-Linked Glycosylation-Dependent and -Independent Mechanisms Regulating CTRP12 Cleavage, Secretion, and Stability

doi: 10.1021/acs.biochem.8b00528

Figure Lengend Snippet: Asn-39 and Asn-297 independently regulate CTRP12 cleavage. (A) Western blot analysis of the cell lysate and supernatant of transfected HEK293 cells expressing wild-type (WT) CTRP12 or the N39Q/N297Q double mutant. (B) Quantification of the ratio of cleaved to full-length (FL) protein (the cleavage index) in the supernatant for WT and the N39Q/N297Q double mutant as shown in panel A. (C) Quantification of the ratio of secreted to intracellular CTRP12 protein (the secretion index) for WT and the N39Q/N297Q double mutant as shown in panel A. (D) A side-by-side comparison of WT, the N39Q single mutant, and the N39Q/N297Q double mutant. (E) Quantification of the ratio of cleaved to full-length (FL) protein (the cleavage index) in the supernatant for WT, N39Q, and N39Q/N297Q as shown in panel D. N = 6 (each lane represents a different sample from an independent transfection). **p < 0.01. ***p < 0.001.

Article Snippet: All C-terminal FLAG epitope-tagged mouse CTRP12 (NCBI GenBank entry {"type":"entrez-protein","attrs":{"text":"NP_080401","term_id":"13385632","term_text":"NP_080401"}} NP_080401 ) single-, double-, and triple-mutant constructs (N39Q, N39A, N287Q, N287A, N297Q, N297A, T41A, S289A, S299A, N39Q/N287Q, N39Q/N297Q, N287Q/N297Q, and N39Q/N287Q/N297Q) were generated by either site-directed mutagenesis or GenScript.

Techniques: Western Blot, Transfection, Expressing, Mutagenesis

Journal: Biochemistry

Article Title: N-Linked Glycosylation-Dependent and -Independent Mechanisms Regulating CTRP12 Cleavage, Secretion, and Stability

doi: 10.1021/acs.biochem.8b00528

Figure Lengend Snippet: CTRP12 double and triple mutants also were not secreted. (A and B) Western blot analysis of the cell lysate and supernatant of transfected HEK293 cells expressing wild-type (WT) CTRP12 or the double (N39Q/N287Q) and triple (N39Q/N287Q/N297Q) mutants. N = 6 (each lane represents a different sample from an independent transfection).

Article Snippet: All C-terminal FLAG epitope-tagged mouse CTRP12 (NCBI GenBank entry {"type":"entrez-protein","attrs":{"text":"NP_080401","term_id":"13385632","term_text":"NP_080401"}} NP_080401 ) single-, double-, and triple-mutant constructs (N39Q, N39A, N287Q, N287A, N297Q, N297A, T41A, S289A, S299A, N39Q/N287Q, N39Q/N297Q, N287Q/N297Q, and N39Q/N287Q/N297Q) were generated by either site-directed mutagenesis or GenScript.

Techniques: Western Blot, Transfection, Expressing

Journal: The FASEB Journal

Article Title: FAM19A1, a brain-enriched and metabolically responsive neurokine, regulates food intake patterns and mouse behaviors

doi: 10.1096/fj.201901232RR

Figure Lengend Snippet: Gene expression of FAM19A1-A5 in human and mouse tissues and in different regions of human and mouse brain. High expression of FAM19A1, FAM19A2, FAM19A4, and FAM19A5 transcript in brain, optic nerve, and spinal cord as measured by TissueScan human major tissue qPCR array (A) and mouse normal tissue qPCR array (B); similar expression patterns of FAM19A1 and FAM19A2 in different brain regions as measured by TissueScan human brain qPCR array (C) and mouse developmental tissue qPCR array (D). The heat maps were generated based on the relative mRNA expression levels in different samples obtained from real-time qPCR analyses.

Article Snippet: C-terminal FLAG epitope-tagged mouse Fam19a1 ( NP_877960 ), Fam19a2 ( NP_001239316 ), Fam19a3 ( NP_899047 ), Fam19a4 ( NP_796207 ), Fam19a5 ( NP_598857 ), as well as the N106A mutant of Fam19a5 , were synthesized by GenScript (Piscataway, NJ, USA) and cloned into the Bam HI and Xho I site of the mammalian expression vector, pcDNA3.1 (+).

Techniques: Gene Expression, Expressing, Generated

Journal: The FASEB Journal

Article Title: FAM19A1, a brain-enriched and metabolically responsive neurokine, regulates food intake patterns and mouse behaviors

doi: 10.1096/fj.201901232RR

Figure Lengend Snippet: Altered expression of Fam19a1-a5 in different mouse brain regions in response to unfed and refed states. Real-time PCR analysis of Fam19a1 (A), Fam19a2 (B), Fam19a3 (C), Fam19a4 (D), and Fam19a5 (E) expression in the cerebellum, cortex, hypothalamus, and hippocampus of mice subjected to being unfed overnight (unfed group; n = 7) or unfed overnight followed by 3 h of refeeding (refed group; n = 8). Expression levels were normalized to β-actin. Data are expressed as means ± sem. Two-tailed Student’s t tests were used for comparing gene expression of unfed vs. refed states in different brain regions. *P < 0.05, **P < 0.01.

Article Snippet: C-terminal FLAG epitope-tagged mouse Fam19a1 ( NP_877960 ), Fam19a2 ( NP_001239316 ), Fam19a3 ( NP_899047 ), Fam19a4 ( NP_796207 ), Fam19a5 ( NP_598857 ), as well as the N106A mutant of Fam19a5 , were synthesized by GenScript (Piscataway, NJ, USA) and cloned into the Bam HI and Xho I site of the mammalian expression vector, pcDNA3.1 (+).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Two Tailed Test, Gene Expression

Journal: The FASEB Journal

Article Title: FAM19A1, a brain-enriched and metabolically responsive neurokine, regulates food intake patterns and mouse behaviors

doi: 10.1096/fj.201901232RR

Figure Lengend Snippet: Generation of Fam19a1 KO mice. A) Schematic showing the gene targeting strategy used to generate Fam19a1 KO mice. B) PCR genotyping results showing the successful generation of WT (+/+), heterozygous (+/−), and homozygous KO (−/−) mice. C) The absence of Fam19a1 mRNA in cortex of KO mice was confirmed by PCR using primer pairs (located in exons 1 and 5) specific for Fam19a1.

Article Snippet: C-terminal FLAG epitope-tagged mouse Fam19a1 ( NP_877960 ), Fam19a2 ( NP_001239316 ), Fam19a3 ( NP_899047 ), Fam19a4 ( NP_796207 ), Fam19a5 ( NP_598857 ), as well as the N106A mutant of Fam19a5 , were synthesized by GenScript (Piscataway, NJ, USA) and cloned into the Bam HI and Xho I site of the mammalian expression vector, pcDNA3.1 (+).

Techniques:

Journal: The FASEB Journal

Article Title: FAM19A1, a brain-enriched and metabolically responsive neurokine, regulates food intake patterns and mouse behaviors

doi: 10.1096/fj.201901232RR

Figure Lengend Snippet: Indirect calorimetry analysis of WT and Fam19a1 KO mice

Article Snippet: C-terminal FLAG epitope-tagged mouse Fam19a1 ( NP_877960 ), Fam19a2 ( NP_001239316 ), Fam19a3 ( NP_899047 ), Fam19a4 ( NP_796207 ), Fam19a5 ( NP_598857 ), as well as the N106A mutant of Fam19a5 , were synthesized by GenScript (Piscataway, NJ, USA) and cloned into the Bam HI and Xho I site of the mammalian expression vector, pcDNA3.1 (+).

Techniques: Activity Assay

Journal: The FASEB Journal

Article Title: FAM19A1, a brain-enriched and metabolically responsive neurokine, regulates food intake patterns and mouse behaviors

doi: 10.1096/fj.201901232RR

Figure Lengend Snippet: Altered food intake patterns and increased physical activity in Fam19a1 KO mice. A–F) Food intake, meal number, and meal size during light and dark cycles in WT and KO male mice (A–C) and female mice (D–F). G, H) Echo-MRI measurements of fat and lean mass in WT and KO mice; 2-tailed Student’s t tests were used for comparing data of WT vs. KO mice. I, J) Physical activity (in the metabolic cage) during light and dark cycle in WT and Fam19a1 KO mice. Two-way ANOVA tests were used for comparing overall activity of WT vs. KO mice. Male mice: WT, n = 9; KO, n = 11. Female mice: WT, n = 9; KO, n = 11. Data are expressed as means ± sem. *P < 0.05, **P < 0.01.

Article Snippet: C-terminal FLAG epitope-tagged mouse Fam19a1 ( NP_877960 ), Fam19a2 ( NP_001239316 ), Fam19a3 ( NP_899047 ), Fam19a4 ( NP_796207 ), Fam19a5 ( NP_598857 ), as well as the N106A mutant of Fam19a5 , were synthesized by GenScript (Piscataway, NJ, USA) and cloned into the Bam HI and Xho I site of the mammalian expression vector, pcDNA3.1 (+).

Techniques: Activity Assay

Journal: The FASEB Journal

Article Title: FAM19A1, a brain-enriched and metabolically responsive neurokine, regulates food intake patterns and mouse behaviors

doi: 10.1096/fj.201901232RR

Figure Lengend Snippet: Reduced anxiety in Fam19a1 KO mice. A) Total activity (2-way ANOVA tests) as well as activity spent in the center and periphery (2-tailed Student’s t tests) in WT and KO mice as measured in open field tests. B) Percentage of time spent in open arms and total distance moved in WT and KO mice as measured by EPM tests (2-tailed Student’s t tests). C) Percentage of inhibition during PPI tests with different prestimulus in male and female WT and Fam19a1 KO mice (2-way ANOVA tests). Male mice: WT, n = 12; KO, n = 7. Female mice: WT, n = 10; KO, n = 11. Data are expressed as means ± sem. **P < 0.01, ***P < 0.001.

Article Snippet: C-terminal FLAG epitope-tagged mouse Fam19a1 ( NP_877960 ), Fam19a2 ( NP_001239316 ), Fam19a3 ( NP_899047 ), Fam19a4 ( NP_796207 ), Fam19a5 ( NP_598857 ), as well as the N106A mutant of Fam19a5 , were synthesized by GenScript (Piscataway, NJ, USA) and cloned into the Bam HI and Xho I site of the mammalian expression vector, pcDNA3.1 (+).

Techniques: Activity Assay, Inhibition

Journal: The FASEB Journal

Article Title: FAM19A1, a brain-enriched and metabolically responsive neurokine, regulates food intake patterns and mouse behaviors

doi: 10.1096/fj.201901232RR

Figure Lengend Snippet: Spatial learning, trace fear conditioning, and hot plate tests in Fam19a1 KO mice. A) Percentage of alternation and total distance moved in WT and KO mice during the Y-maze spontaneous alternation tests (2-tailed Student’s t tests). B) Percentage of freezing in training (2-way ANOVA tests), cue test, and context test (2-tailed Student’s t tests) in WT and KO mice during trace fear conditioning tests. C) Time of latency in male and female WT and Fam19a1 KO mice during hot plate test (2-tailed Student’s t tests). Male mice: WT, n = 12; KO, n = 7. Female mice: WT, n = 9; KO, n = 11. Data are expressed as means ± sem. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: C-terminal FLAG epitope-tagged mouse Fam19a1 ( NP_877960 ), Fam19a2 ( NP_001239316 ), Fam19a3 ( NP_899047 ), Fam19a4 ( NP_796207 ), Fam19a5 ( NP_598857 ), as well as the N106A mutant of Fam19a5 , were synthesized by GenScript (Piscataway, NJ, USA) and cloned into the Bam HI and Xho I site of the mammalian expression vector, pcDNA3.1 (+).

Techniques: Hot Plate Test

Journal: The FASEB Journal

Article Title: FAM19A1, a brain-enriched and metabolically responsive neurokine, regulates food intake patterns and mouse behaviors

doi: 10.1096/fj.201901232RR

Figure Lengend Snippet: Increased dopamine turnover in the striatum in female Fam19a1 KO mice. A–G) Monoamines and their metabolites [NE (A), dopamine (B, DA), 3,4-dihydroxyphenylacetic acid (C, DOPAC), 3-methoxytyramine (D, 3-MT), homovanillic acid (E, HVA), 5-hydroxytryptamine (F, 5-HT; serotonin), and 5-HIAA (G)] concentrations in the striatum were measured by HPLC with electrochemical detection. H, I) Dopamine turnover rate (the ratio of the dopamine metabolites to dopamine. J) Serotonin turnover rate (the ratio of serotonin metabolite 5-HIAA to 5-HT). WT, n = 8; KO, n = 9. Data are expressed as means ± sem. Two-tailed Student’s t tests were used for comparing data of WT vs. KO mice. *P < 0.05, ***P < 0.001.

Article Snippet: C-terminal FLAG epitope-tagged mouse Fam19a1 ( NP_877960 ), Fam19a2 ( NP_001239316 ), Fam19a3 ( NP_899047 ), Fam19a4 ( NP_796207 ), Fam19a5 ( NP_598857 ), as well as the N106A mutant of Fam19a5 , were synthesized by GenScript (Piscataway, NJ, USA) and cloned into the Bam HI and Xho I site of the mammalian expression vector, pcDNA3.1 (+).

Techniques: Two Tailed Test