Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: LRRC8A anion channels modulate vascular reactivity via association with Myosin Phosphatase Rho Interacting Protein (MPRIP)

doi: 10.1096/fj.202300561R

Figure Lengend Snippet: A) Immunoprecipitation (IP) of endogenous LRRC8A from VSMCs confirms the presence of LRRC8A in the whole cell (WC) lysate and precipitated protein from WT but not LRRC8A KO cells. MPRIP is present in the WC lysate from both genotypes but is only pulled down in LRRC8A WT cells. B) Proximity Ligation Assay (PLA) signal from α-MPRIP/α-LRRC8A in WT VSMCs. Small white spots represent PLA signal, the larger spots are nuclei. Counts of spots/nucleus from multiple fields of confluent WT and LRRC8A KO VSMCs (20x). PLA spots were smaller and significantly less numerous in KO cells. The bottom image shows red PLA spots with green nuclei as a side-view of cells. C) Confocal images of HEK293T cells co-expressing LRRC8A-meGFP and MPRIP-mCherry. Note co-localization at the plasma membrane, particularly at ruffled borders but not in LRRC8A-containing intracellular vesicles. D) Sites of protein binding to MPRIP. The first Plextrin Homology (PH) domain (aa 44–152) binds actin. C-terminal coiled-coil domains mediate interaction with RhoA and MYPT1 (M). Below the map are the constructs utilized to determine the site of MPRIP binding to LRRC8A (8A) was localized to aa 389–545. Vertical grey lines represent specific cysteines that can be oxidized based upon the Oxymouse database (Cys103, 120, 235, 361, 506, 571, 723, 830). Susceptible cysteines are present in the binding regions for all 4 protein partners. E) Immunoprecipitation of MPRIP-DDK fragments by full length LRRC8A-Myc in HEK293T cells expressing these constructs. Arrows identify overexpressed proteins, or the site where they would be expected to run (see IP: α-Myc) in whole cell lysates (WCL, left) and in immunoprecipitated protein (IP, right). All C-terminal MPRIP deletion mutants associate with LRRC8A, but the C-terminal MPRIP fragment 879–1038 does not associate with LRRC8A. The 213–545 peptide associated with LRRC8A as did the region between 389 and 545 which contains PH2 (see IP: α-LRRC8A).

Article Snippet: A C-terminal tagged clone of human MPRIP (DDK (Flag), Genscript, Cat # OHu00028D) was truncated by insertion of an additional Flag tag followed by a premature stop codon using the QuickChange site-directed mutagenesis kit (Agilent).

Techniques: Immunoprecipitation, Proximity Ligation Assay, Expressing, Clinical Proteomics, Membrane, Protein Binding, Construct, Binding 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: 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