Journal: Cell

Article Title: The hyaluronidase, TMEM2, promotes ER homeostasis and longevity independent of the UPR ER

doi: 10.1016/j.cell.2019.10.018

Figure Lengend Snippet: Whole genome CRISPR-KO library screen identifies TMEM2 as a potent modulator of ER stress sensitivity. A) Screen outline: Human immortalized fibroblasts were transduced with a genome-wide sgRNA lentiviral library and cultured for two weeks to maximize genome editing and target protein depletion. Cells were then split into control and Tunicamycin treatment and harvested after 3 weeks for sequencing (as described in detail in STAR METHODS) B) Comparison of gene depletion p-values between control and Tunicamycin-treated cells (individual depletion/enrichment are available in Supplemental Table 1) C) Enrichment analysis of the top differentially depleted genes (using 10% FDR as a cutoff) using the EnrichR online tool (https://amp.pharm.mssm.edu/Enrichr/). D) Viability and proliferation of Wildtype and clonal TMEM2-KO human immortalized fibroblast in the presence of Tunicamycin-induced ER stress with or without CMV-TMEM2 overexpression. Results are relative to untreated control to adjust for variability in initial cell number between cell lines. Cell density at the endpoint of a 5 day treatment period was determined via CellTiter-Glo analysis (CTG) (as described in detail in STAR METHODS); (n=3). Statistical Analysis: One-way ANOVA analysis with post-hoc Bonferroni-Holm analysis

Article Snippet: Human foreskin fibroblast, BJ , ATCC , CRL-2522.

Techniques: CRISPR, Transduction, Genome Wide, Cell Culture, Control, Sequencing, Comparison, Over Expression

Journal: Cell

Article Title: The hyaluronidase, TMEM2, promotes ER homeostasis and longevity independent of the UPR ER

doi: 10.1016/j.cell.2019.10.018

Figure Lengend Snippet: hTMEM2 overexpression promotes immunity and extends lifespan through mpk-1/pmk-1. A) Lifespans were measured in Wildtype and sur-5p::hsf-1 animals on EV, jnk-1, mpk-1, or pmk-1 RNAi from hatch. Data is representative of three independent trials. B) Fluorescent micrographs of Wildtype (N2) and sur-5p::hTMEM2 animals expressing the immune response reporter, T24B8.5p::GFP. Animals were grown on EV or pmk-1 RNAi as described in STAR METHODS. Data is representative of three independent trials. C) Survival was scored in Wildtype (N2) and sur-5p::hTMEM2 animals exposed to PA14 infection at L4. Survival was scored every 6 hours as described in STAR METHODS. Data is representative of two independent trials. D) Lifespans were measured in Wildtype (N2) and sur-5p::hTMEM2 animals grown on dead EV RNAi from hatch. Bacteria were killed by UV irradiation, as described in STAR METHODS. All statistics for C-D were performed using Log-Rank (Mantel-Cox) test using PRISM, and are available in Supplemental Table 2. E) Wildtype and CMV-TMEM2 overexpressing human fibroblasts were exposed to lipopolysaccharides (LPS) derived from the E. coli bacteria strain (O111:B4). Resistance to the presence of LPS was measured through CTG to determine the cell density after 5 days of exposure; (n=3). Statistical analysis: One-way ANOVA test with a post-hoc Bonferroni-Holm analysis

Article Snippet: Human foreskin fibroblast, BJ , ATCC , CRL-2522.

Techniques: Over Expression, Expressing, Infection, Bacteria, Irradiation, Derivative Assay

Journal: Cell

Article Title: The hyaluronidase, TMEM2, promotes ER homeostasis and longevity independent of the UPR ER

doi: 10.1016/j.cell.2019.10.018

Figure Lengend Snippet: Lifespan extension through canonical xbp-1s signaling is not dependent on mpk-1/pmk-1 and is distinct from hTMEM2. A) Lifespans were measured in Wildtype (N2) and rab-3p::xbp-1s animals on EV, jnk-1, mpk-1, or pmk-1 RNAi from hatch. Data is representative of three independent trials. B) Lifespans were measured in Wildtype (N2) and rab-3p::xbp-1s animals grown on dead EV RNAi from hatch as per 5D. Data is representative of two independent trials. C-D) Lifespans were measured in Wildtype (N2), sur-5p::hTMEM2, rab-3p::xbp-1s, and sur-5p::hTMEM2/rab-3p::xbp-1s animals in the absence (C) and presence (D) of FUDR. Animals were grown on EV RNAi from hatch, and the assay was either performed on standard EV plates (C) or moved to FUDR containing plates at L4 for (D) - see STAR Methods for details. E) Graphical representation of the key insight generated by this work. In human fibroblasts, TMEM2 breaks down HMW-HA into LMW-HA within the ECM. Through interaction with CD44, LMW-HA influences ERK/mpk1- and p38/pmk-1-mediated MAPK-signaling. This in turn alters ER stress resistance and pathogen resistance in human fibroblasts. In C. elegans, TMEM2-mediated changes to glycosaminoglycan (GAG) metabolism cause a shift in MAPK signaling. This in turn alters the response to ER stress and with it, changes pathogen resistance and the lifespan of the animals.

Article Snippet: Human foreskin fibroblast, BJ , ATCC , CRL-2522.

Techniques: Generated

Journal: Cell

Article Title: The hyaluronidase, TMEM2, promotes ER homeostasis and longevity independent of the UPR ER

doi: 10.1016/j.cell.2019.10.018

Figure Lengend Snippet: TMEM2’s enzymatic breakdown of HMW-HA to LMW-HA is responsible for the ER stress phenotype. A) The CMV-TMEM2 plasmid was altered through site-directed mutagenesis in order to disrupt HAase enzymatic activity of the gene. ER stress resistance was then measured through CTG analysis. The ER stress resistance was then compared between the constructs with no or a neutral mutation (ΔD275N) of the gene, and two lines in which the HAase function of TMEM2 was diminished (ΔP265C; ΔD273N); (n=3). B) Resistance to Tunicamycin-induced ER stress was measured in Wildtype and TMEM2-KO human fibroblasts in the presence and absence of supplemented hyaluronidase (HAase) (Concentration in bar graph: HAase 5U/ml). All HAase concentrations tested (0.6U/ml – 160U/ml) were equally able to evoke this phenotype. C-D) Wildtype and TMEM2-KO cells were exposed to low molecular weight hyaluronan (LMW-HA; <20kDa in molecular weight) medium molecular weight hyaluronan (MMW-HA; 200–1000kDa) or high molecular weight hyaluronan (HMW-HA; >1000kDa). The concentration of LMW-HA, MMW-HA and HMW-HA in the bar graphs was limited to 600ng/ml, since HMW-HA did not go fully into solution at higher concentrations (marked with #).

Article Snippet: Human foreskin fibroblast, BJ , ATCC , CRL-2522.

Techniques: Plasmid Preparation, Mutagenesis, Activity Assay, Construct, Concentration Assay, Molecular Weight, High Molecular Weight

Journal: Cell

Article Title: The hyaluronidase, TMEM2, promotes ER homeostasis and longevity independent of the UPR ER

doi: 10.1016/j.cell.2019.10.018

Figure Lengend Snippet: EXPERIMENTAL MODEL AND SUBJECT DETAILS

Article Snippet: Human foreskin fibroblast, BJ , ATCC , CRL-2522.

Techniques: Virus, Recombinant, Cell Viability Assay, Enzyme-linked Immunosorbent Assay, Bradford Assay, Gel Extraction, Purification, Software

Journal: iScience

Article Title: Neonatal intake of Omega-3 fatty acids enhances lipid oxidation in adipocyte precursors

doi: 10.1016/j.isci.2022.105750

Figure Lengend Snippet: Bulk RNA-seq of flow-sorted APC CD24 + progenitors and CD24 − preadipocytes reveals transcriptomic signatures for adipogenic, mitochondrial, and energetics pathways by low n6/n3 FA ratios (A) Differential gene expression by n6/n3 exposure group within either progenitor or preadipocyte APC subtypes was analyzed using comparative analysis in Ingenuity Pathway Analysis. Significantly enriched canonical pathways in common between CD24 + progenitor and CD24 − preadipocyte APCs were ordered by FDR p value (≤0.01) and colored by the predicted activation Z score. Coloring indicates common pathways predicted to be “on” (yellow), “off” (navy), or “no prediction” (white) due to low n6/n3 exposure. Pathway activation Z score values are presented for either APC subtype, and values ≥2.0 or ≤ −2.0 are considered significant. (B) Significantly different genes in either CD24 + progenitor or CD24 − preadipocyte relative to the high n6/n3 FA ratio group colored by Log2 fold change and grouped into transcription factor genes (Regulatory) and signal transduction pathway genes (Ligands/Kinases). (C) Significantly different lipid metabolism, mitochondrial, and glycolytic genes indicating that the low n6/n3 FA ratio changes oxidative capacity potentially through aldehyde dehydrogenases (Aldh1a1 and Aldh1a3), uncoupling protein 2 (Ucp2), citrate synthase (CS), isocitrate dehydrogenase (Idh1 and 2), malic enzyme (Me1), malate dehydrogenase (Mdh1), phosphofructokinases (Pfkm, Pfkp, and Pfkfb1), and transketolase (Tk) (See also Figure S2 ). (D) Canonical Pathway enrichment within either CD24 + progenitor or CD24 − preadipocyte APCs plotted by the percentage DE-Gs within a pathway (%) by the pathway enrichment significance (-log(p value)) and colored by the predicted pathway activation ( Z score, >2.0 and < −2.0 was considered significant) (See also for all pathways). (E) Immunoblots for NR2F2, UCP2, C/EBPα, and DNMT1 from all lineage negative, CD29+/CD34+, and Sca1+ APCs isolated by flow cytometry from pooled litters (n = 3 litter dyads per n6/n3 FA ratio group; litters standardized to 6-8 pups/litter). (F) EVC005 murine APC cell line was treated with and without NR2F2 ligand 1-DSO for 48 h, which induced gene expression of Prdm16, P2rx5, Runx1, Ucp2, Dnmt1, Pparγ2, and C/ebpα. (G) TMRE live cell mitochondrial potential dye following 48 h of 1-DSO treatment in EVC005 cells, indicating an increased mitochondrial potential. Panels F and G are n = 3 wells per treatment group. Scale bar represents 50 μm. Data are represented as mean ± SEM.

Article Snippet: Ucp2 mouse TaqMan Primer , ThermoFisher Scientific , Cat# Mm00627599_m1.

Techniques: RNA Sequencing, Gene Expression, Activation Assay, Transduction, Western Blot, Isolation, Flow Cytometry

Journal: iScience

Article Title: Neonatal intake of Omega-3 fatty acids enhances lipid oxidation in adipocyte precursors

doi: 10.1016/j.isci.2022.105750

Figure Lengend Snippet:

Article Snippet: Ucp2 mouse TaqMan Primer , ThermoFisher Scientific , Cat# Mm00627599_m1.

Techniques: Recombinant, High Molecular Weight, Enzyme-linked Immunosorbent Assay, Software, Modification

Journal: Biomedicines

Article Title: Renalase Overexpression-Mediated Excessive Metabolism of Peripheral Dopamine, DOPAL Accumulation, and α-Synuclein Aggregation in Baroreflex Afferents Contribute to Neuronal Degeneration and Autonomic Dysfunction.

doi: 10.3390/biomedicines13051243

Figure Lengend Snippet: Figure 2. Expression of renalase in rats was increased after 6-OHDA treatment. (A) Detection of serum renalase concentration from the 2nd to 5th week after 6-OHDA application by ELISA kit (n = 6 rats per group). (B) qRT-PCR detection of relative renalase expression in NG, NTS, and heart tissues of Parkinson’s rats and sham group (4th week after 6-OHDA application, n = 5 rats per group). (C,D) Western blot assay showing relative protein levels of renalase in NG, NTS, and heart tissues of Parkinson’s rats and sham group (n = 4 rats per group). Data are presented as mean ± SD, * p < 0.05 and ** p < 0.01 vs. sham.

Article Snippet: The concentration of RNLS was detected using the Rat RNLS ELISA Kit (CSB-EL002965RA, Cusabio, Wuhan, China) and the O.D. value was measured at 450 nm.

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot

Journal: Biomedicines

Article Title: Renalase Overexpression-Mediated Excessive Metabolism of Peripheral Dopamine, DOPAL Accumulation, and α-Synuclein Aggregation in Baroreflex Afferents Contribute to Neuronal Degeneration and Autonomic Dysfunction.

doi: 10.3390/biomedicines13051243

Figure Lengend Snippet: Figure 3. PC12 cells transfected with RNLS overexpression plasmid (or empty vector control) continued to overexpress and metabolize DA. (A,B) Transient transfection of PC12 cells with RNLS overexpression plasmid (or empty vector control) expressing the protein. Western blot assay showing relative protein levels of RNLS in PC12 cells at different time points (n = 6). (C) qRT-PCR detection of relative RNLS at different time points after PC12 cells transfected with (n = 6). (D) Detection of relative dopamine (DA) concentration in PC12 cells at different time points by ELISA kit following RNLS overexpression or NC, showing altered DA levels (n = 6). Data are presented as mean ± SD, * p < 0.05 and ** p < 0.01 vs. Ctrl.

Article Snippet: The concentration of RNLS was detected using the Rat RNLS ELISA Kit (CSB-EL002965RA, Cusabio, Wuhan, China) and the O.D. value was measured at 450 nm.

Techniques: Transfection, Over Expression, Plasmid Preparation, Control, Expressing, Western Blot, Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Biomedicines

Article Title: Renalase Overexpression-Mediated Excessive Metabolism of Peripheral Dopamine, DOPAL Accumulation, and α-Synuclein Aggregation in Baroreflex Afferents Contribute to Neuronal Degeneration and Autonomic Dysfunction.

doi: 10.3390/biomedicines13051243

Figure Lengend Snippet: Figure 4. Increased levels of DA metabolites after RNLS overexpression of in PC12 cells. (A,B) Detection of DOPAL content at different time points in PC12 cells after RNLS overexpres- sion by high performance liquid chromatography (n = 3). (C) qRT-PCR detection of relative ALDH expression at different time points after PC12 cells were transfected with (n = 6). (D,E) Western blot analysis showing relative protein levels of ALDH at different time points after PC12 cells were transfected with (n = 6). Data are presented as mean ± SD, * p< 0.05 and ** p < 0.01 vs. Ctrl.

Article Snippet: The concentration of RNLS was detected using the Rat RNLS ELISA Kit (CSB-EL002965RA, Cusabio, Wuhan, China) and the O.D. value was measured at 450 nm.

Techniques: Over Expression, High Performance Liquid Chromatography, Quantitative RT-PCR, Expressing, Transfection, Western Blot

Journal: Biomedicines

Article Title: Renalase Overexpression-Mediated Excessive Metabolism of Peripheral Dopamine, DOPAL Accumulation, and α-Synuclein Aggregation in Baroreflex Afferents Contribute to Neuronal Degeneration and Autonomic Dysfunction.

doi: 10.3390/biomedicines13051243

Figure Lengend Snippet: Figure 6. α-Syn aggregated in PC12 cells transfected with RNLS for 96 h. (A,B) Representative graph of Tri-αS and higher molecular weight α-Syn aggregates in PC12 cells transfected with RNLS for 96 h (n = 6). (C(a,b)) Immunofluorescent photomicrographs of transfected cells (overexpressing RNLS protein for 96 h), cytoplasmic aggregates in the cell neurotic processes are highlighted by white arrows in the figure. (C(c,d)) Untreated control PC12 cells. Scale bar: 20 µm. Data are presented as mean ± SD, ** p < 0.01 vs. Ctrl.

Article Snippet: The concentration of RNLS was detected using the Rat RNLS ELISA Kit (CSB-EL002965RA, Cusabio, Wuhan, China) and the O.D. value was measured at 450 nm.

Techniques: Transfection, Molecular Weight, Control

Journal: Biomedicines

Article Title: Renalase Overexpression-Mediated Excessive Metabolism of Peripheral Dopamine, DOPAL Accumulation, and α-Synuclein Aggregation in Baroreflex Afferents Contribute to Neuronal Degeneration and Autonomic Dysfunction.

doi: 10.3390/biomedicines13051243

Figure Lengend Snippet: Figure 5. Changes in α-Syn content at different time points in RNLS-overexpressing PC12 cells. (A) qRT-PCR detection of relative α-Syn expression at different time points after PC12 cells trans- fected with RNLS (n = 6). (B) Representative image of immunofluorescence with enhanced α-Syn fluorescence after transfection of PC12 cells. Scale bar: 20 µm. (C) Immunohistochemical quantitative statistics (n = 60). (D–G) Western blot analysis showing relative protein levels of monomer of α-Syn (Mon-αS) at different time points after PC12 cells were transfected with RNLS (n = 6). (E–H) Western blot analysis showing relative protein levels of dimer of α-Syn (Dim-αS) at different time points after PC12 cells were transfected with RNLS (n = 6). (F–I) Western blot analysis showing relative protein levels of trimer of α-Syn (Tri-αS) at different time points after PC12 cells were transfected with RNLS (n = 6). Data are presented as mean ± SD, * p < 0.05 and ** p < 0.01 vs. Ctrl.

Article Snippet: The concentration of RNLS was detected using the Rat RNLS ELISA Kit (CSB-EL002965RA, Cusabio, Wuhan, China) and the O.D. value was measured at 450 nm.

Techniques: Quantitative RT-PCR, Expressing, Immunofluorescence, Fluorescence, Transfection, Immunohistochemical staining, Western Blot

Journal: Biomedicines

Article Title: Renalase Overexpression-Mediated Excessive Metabolism of Peripheral Dopamine, DOPAL Accumulation, and α-Synuclein Aggregation in Baroreflex Afferents Contribute to Neuronal Degeneration and Autonomic Dysfunction.

doi: 10.3390/biomedicines13051243

Figure Lengend Snippet: Figure 7. The content of tyrosine hydrolase (TH) decreased after PC12 cells were transfected with RNLS. (A) qRT-PCR detection of relative TH expression at different time points after PC12 cells transfected with RNLS (n = 6). (B,C) Western blot analysis showing relative protein levels of TH at different time points after PC12 cells were transfected withR NLS (n = 6). (D) Representative graph of immunofluorescent (IF) results of TH in PC12 cells at different time points administered with RNLS overexpression. Scale bar: 20 µm. (E) Immunohistochemical quantitative statistics (n = 60). Data are presented as mean ± SD, * p < 0.05 and ** p < 0.01 vs. Ctrl.

Article Snippet: The concentration of RNLS was detected using the Rat RNLS ELISA Kit (CSB-EL002965RA, Cusabio, Wuhan, China) and the O.D. value was measured at 450 nm.

Techniques: Transfection, Quantitative RT-PCR, Expressing, Western Blot, Over Expression, Immunohistochemical staining

Journal: Biomedicines

Article Title: Renalase Overexpression-Mediated Excessive Metabolism of Peripheral Dopamine, DOPAL Accumulation, and α-Synuclein Aggregation in Baroreflex Afferents Contribute to Neuronal Degeneration and Autonomic Dysfunction.

doi: 10.3390/biomedicines13051243

Figure Lengend Snippet: Figure 8. Cell viability decreased after PC12 cells were transfected with RNLS. (A) Live/dead cell viability assay of cultured PC12 cells. The cells were transfected with plasmids and cultured for 24, 48, 72, or 96 h and then stained with the Calcein-AM/PI Double Staining Kit. The live and dead cells exhibited green and red fluorescence. Scale bar: 10 µm. (B) Immunohistochemical quantitative statistics (n = 3). (C) CCK-8 was used to determine PC12 cells viability following overexpression or NC for 24, 48, 72 or 96 h (n = 12). Data are presented as mean ± SD, ** p < 0.01 vs. Ctrl.

Article Snippet: The concentration of RNLS was detected using the Rat RNLS ELISA Kit (CSB-EL002965RA, Cusabio, Wuhan, China) and the O.D. value was measured at 450 nm.

Techniques: Transfection, Viability Assay, Cell Culture, Staining, Double Staining, Fluorescence, Immunohistochemical staining, CCK-8 Assay, Over Expression

Journal: Biomedicines

Article Title: Renalase Overexpression-Mediated Excessive Metabolism of Peripheral Dopamine, DOPAL Accumulation, and α-Synuclein Aggregation in Baroreflex Afferents Contribute to Neuronal Degeneration and Autonomic Dysfunction.

doi: 10.3390/biomedicines13051243

Figure Lengend Snippet: Figure 9. Decreased expression of axonal transporters after RNLS overexpression in PC12 cells. (A) qRT-PCR detection of relative dynein (DYN) expression at different time points after PC12 cells transfected with RNLS (n = 6). (B,C) Western blot analysis showing relative protein levels of dynein at different time points after PC12 cells were transfected with RNLS (n = 6). (D) mRNA expression of relative kinesin in PC12 cells at different time points (n = 6). (E,F) Western blot analysis showing relative protein levels of KHC at different time points after PC12 cells were transfected with RNLS (n = 6). Data are presented as mean ± SD, * p < 0.05 and ** p < 0.01 vs. Ctrl.

Article Snippet: The concentration of RNLS was detected using the Rat RNLS ELISA Kit (CSB-EL002965RA, Cusabio, Wuhan, China) and the O.D. value was measured at 450 nm.

Techniques: Expressing, Over Expression, Quantitative RT-PCR, Transfection, Western Blot

Journal: Biomedicines

Article Title: Renalase Overexpression-Mediated Excessive Metabolism of Peripheral Dopamine, DOPAL Accumulation, and α-Synuclein Aggregation in Baroreflex Afferents Contribute to Neuronal Degeneration and Autonomic Dysfunction.

doi: 10.3390/biomedicines13051243

Figure Lengend Snippet: Figure 10. Ultrastructural damage after RNLS overexpression in PC12 cells. (A,B) The relative ∆Ψ m in PC12 cells for each treatment at different time points, as determined by JC-1 staining (n = 15). Data are presented as mean ± SD, ** p < 0.01 vs. Ctrl; scale bar: 20 µm; ∆Ψm: mitochondrial membrane potential. (C) Representative images of transmission microscopy at different time points after PC12 cells were transfected with RNLS. Scale bar: 1 µm and 2 µm, direct magnification: ×1200, ×2000 and ×4000, the red arrow indicates mitochondria.

Article Snippet: The concentration of RNLS was detected using the Rat RNLS ELISA Kit (CSB-EL002965RA, Cusabio, Wuhan, China) and the O.D. value was measured at 450 nm.

Techniques: Over Expression, Staining, Membrane, Transmission Assay, Microscopy, Transfection

Journal: Biomedicines

Article Title: Renalase Overexpression-Mediated Excessive Metabolism of Peripheral Dopamine, DOPAL Accumulation, and α-Synuclein Aggregation in Baroreflex Afferents Contribute to Neuronal Degeneration and Autonomic Dysfunction.

doi: 10.3390/biomedicines13051243

Figure Lengend Snippet: Figure 11. Numerous clues to PD peripheral pathology. (A) Vagal application of DOPAL (3,4- dihydroxyphenylacetaldehyde) to simulate PD-like autonomic dysfunction strongly suggests that DOPAL will likely induce the expression and aggregation of α-Syn/oligomers at the injection site, which will then be transported to the heart and the NG. Axonal transportation is likely to be impaired by DOPAL and DOPAL-mediated oligomer α-Syn [22]. (B) Typical PD model causes the RNLS up-regulation. (C) The pathological α-Syn protein fibers were injected into the mouse intestine, and it was found that the α-Syn protein eventually diffused into the substantia nigra pars compacta, where it degraded dopaminergic neurons. (D) Orthostatic hypotension (OH) has been linked to a higher risk of Parkinson’s disease in studies [48,49]. OH is a predictor of motor decline in individuals with early PD [50]. The experiment confirmed that the change in BP also occurred before the movement disorder in rats treated with 6-OHDA. (E) Current mechanisms and central nervous system origins of PD: MAO-mediated DA metabolism in the central nervous system. (F) The role of RNLS in peripheral etiology of PD.

Article Snippet: The concentration of RNLS was detected using the Rat RNLS ELISA Kit (CSB-EL002965RA, Cusabio, Wuhan, China) and the O.D. value was measured at 450 nm.

Techniques: Expressing, Injection

Journal: PLoS Genetics

Article Title: Astakine 2—the Dark Knight Linking Melatonin to Circadian Regulation in Crustaceans

doi: 10.1371/journal.pgen.1003361

Figure Lengend Snippet: A) In vitro GST pull-down assay of His-Trx- Pl BMAL1 by GST- Pl RACK1. The elution fractions of the GST- Pl RACK1 pull-down assay were examined by western blot analysis using anti-GST and anti-His antibodies. Lanes 1 and 5: elution fraction of GST- Pl RACK1 pull-down of HisTrx- Pl BMAL1; Lanes 2 and 6: GST- Pl RACK1 pull-down of His-Trx; Lanes 3 and 7: GST pull-down of HisTrx- Pl BMAL1; Lanes 4 and 8; GST pull-down of His-Trx. B) The protein-protein interaction of Pl RACK1 and Pl BMAL1 was analyzed by far western blotting. C) Binding of AST2 and Pl BMAL1 to GST- Pl RACK1. GST- Pl RACK1 (0, 5, 50 or 500 ng) was bound to Glutathione Sepharose beads and incubated with 500 ng His-Trx- Pl BMAL1 and 500 ng His-Trx-AST2. Bound proteins were eluted and immunoblotted for Pl BMAL1 and AST2 using an anti-His antibody and for Pl RACK1 with an anti-GST antibody.

Article Snippet: A goat polyclonal antibody to human BMAL1 (N-5: SC-8550) and goat polyclonal antibody to mouse CLK (S-19: SC-6927) were obtained from Santa Cruz Biotechnology.

Techniques: In Vitro, Pull Down Assay, Western Blot, Far Western Blot, Binding Assay, Incubation

Journal: PLoS Genetics

Article Title: Astakine 2—the Dark Knight Linking Melatonin to Circadian Regulation in Crustaceans

doi: 10.1371/journal.pgen.1003361

Figure Lengend Snippet: A) Relative amounts of AST2, Pl RACK1 and Pl BMAL1 proteins in brain extracts as determined by ELISA at 3 h after light turned off or on. B) Relative amounts of AST2, Pl RACK1 and Pl BMAL1 proteins in HPT extracts as determined by ELISA at 3 h after light turned off or on. The asterisks indicate significant differences (*P<0.05); one-way ANOVA with Duncan's new multiple-range test and the Tukey test. Results are representative of three independent experiments. Error bars indicate SD from three replicates and the experiment has been repeated three times with similar results. C) Immunoprecipitation (IP) of brain extract using antibodies against RACK1, BMAL1 and AST2 revealed that a Pl RACK1-AST2 complex was present in the brain (B) during the day. D) Immunoprecipitation (IP) of brain extract using antibodies against RACK1, BMAL1 and AST2 revealed the presence of a high-molecular-weight complex (approximately 200 kDa) composed of all three proteins in the brain (B) at night. C–D) The immunoprecipitated complex was analyzed by western blotting (WB) using another antibody. “B” and “HPT” represent brain and hematopoietic tissue, respectively. The antibodies used for immunoprecipitation (IP) and detection (WB) is indicated at the top and bottom of the blots, respectively, and +DTT and −DTT represent reducing and non-reducing conditions, respectively. Molecular masses are indicated at the left.

Article Snippet: A goat polyclonal antibody to human BMAL1 (N-5: SC-8550) and goat polyclonal antibody to mouse CLK (S-19: SC-6927) were obtained from Santa Cruz Biotechnology.

Techniques: Enzyme-linked Immunosorbent Assay, Immunoprecipitation, High Molecular Weight, Western Blot

Journal: PLoS Genetics

Article Title: Astakine 2—the Dark Knight Linking Melatonin to Circadian Regulation in Crustaceans

doi: 10.1371/journal.pgen.1003361

Figure Lengend Snippet: A) Immunoprecipitation (IP) of endogenous CLOCK (CLK) and Pl BMAL1 in the brain (B) and HPT. Total proteins were extracted from the brain and HPT and were immunoprecipitated (IP) with the antibodies (Ab) indicated on the top of the blots, followed by western blot (WB) detection with antibodies against CLK or BMAL1 as shown at the bottoms of the blots. Reducing and non-reducing conditions of the samples are indicated by +DTT or −DTT, respectively. B) The levels of the CLK- Pl BMAL1 and AST2- Pl RACK1- Pl BMAL1 protein complexes were analyzed by SDS-PAGE under non-reducing conditions (sample without DTT) followed by western blotting using an antibody against BMAL1. The AST2- Pl RACK1 heterodimer and CLK were detected by western blotting using antibodies against AST2 and CLK, respectively. Time points were taken at 03:00, 06:00, 12:00, 18:00, and 21:00 (n = 4). An actin protein was used as an internal control. The horizontal band at the top of the histogram indicates the light condition (white = light, black = dark). C) Relative amounts of CLK- Pl BMAL1 protein complex in brain extracts at different time points (n = 3) as determined by western blotting. D) Relative amounts of AST2- Pl RACK1- Pl BMAL1 protein complex in brain extracts at different time points (n = 3) as determined by western blotting. E) Relative amounts of AST2- Pl RACK1 protein complex in brain extracts at different time points (n = 3) as determined by western blotting. F) Relative amounts of CLK protein in brain extracts at different time points (n = 3) as determined by western blotting. Average protein level in Graphs C–F was quantitated using Quantity One. The asterisks indicate significant differences (*P<0.05, **P<0.01); one-way ANOVA with Duncan's new multiple-range test and the Tukey test. Results are representative of three independent experiments. Error bars indicate SD from three replicates and the experiment has been repeated three times with similar results. G) Melatonin injection inhibited the formation of the CLK- Pl BMAL1 complex; this inhibition is mediated by the AST2- Pl RACK1- Pl BMAL1 complex. The levels of the CLK- Pl BMAL1 and AST2- Pl RACK1- Pl BMAL1 protein complexes were analyzed by SDS-PAGE under non-reducing conditions (sample without DTT) followed by western blotting using an antibody against BMAL1. The level of β-actin was used as an internal control. H) Relative levels of CLK- Pl BMAL1 and AST2- Pl RACK1- Pl BMAL1 complexes in vivo , in the brain of crayfish after injection of melatonin and then brain extracts were analyzed by western blotting using an antibody against BMAL1. Grey bars = melatonin (4.3 nmol/g), white bars = control injection (PBS). I) Relative levels of CLK- Pl BMAL1 and AST2- Pl RACK1- Pl BMAL1 complexes in vivo , in the HPT of crayfish after injection of melatonin and then HPT extracts were analyzed by western blotting using an antibody against BMAL1. Grey bars = melatonin (4.3 nmol/g), white bars = control injection (PBS). The level of β-actin was used as an internal control. Asterisks indicate significant differences (* P <0.05, ** P <0.01). Quantity One analysis was used to quantify the intensity of protein bands. Graphs (H and I) represent the quantification of each complex formation, using Quantity one. Results are representative of three independent experiments. Statistical significance: *P<0.05, **P<0.01 using Student's paired t-test (error bars indicate SD from nine replicates).

Article Snippet: A goat polyclonal antibody to human BMAL1 (N-5: SC-8550) and goat polyclonal antibody to mouse CLK (S-19: SC-6927) were obtained from Santa Cruz Biotechnology.

Techniques: Immunoprecipitation, Western Blot, SDS Page, Control, Injection, Inhibition, In Vivo

Journal: PLoS Genetics

Article Title: Astakine 2—the Dark Knight Linking Melatonin to Circadian Regulation in Crustaceans

doi: 10.1371/journal.pgen.1003361

Figure Lengend Snippet: A) The relative expression levels of AST2 in the brain at 03:00, 06:00, 12:00, and 21:00 (n = 9) after partial knock down of AST2 (grey) in the brain by dsRNA injection and detection by qPCR; dsGFP injection was used as a control (white). B) The relative expression levels of AST2 in hemocytes at 03:00, 06:00, 12:00, and 21:00 (n = 9) after partial knock down of AST2 (grey) by dsRNA injection and detection by qPCR; dsGFP injection was used as a control (white). Graph A and B represent AST2 mRNA levels, using qPCR. Results are representative of three independent experiments. Error bars indicate SD of nine replicates. The asterisks indicate significant differences (*P<0.05, **P<0.01); Student's paired t-test. C) The effect of dsAST2 on complex formation in the brain in vivo was examined by western blotting of brain extracts and detection as follows: for the CLK- Pl BMAL1 complex an anti-BMAL1 antibody, for the AST2- Pl RACK1- Pl BMAL1 complex an anti-BMAL1 antibody, and for the AST2- Pl RACK1 an anti- AST2 antibody was used. The horizontal band at the top of the histogram indicates the light condition (white = light, black = dark). D) Relative amounts of CLK- Pl BMAL1 protein complex in the brain in vivo was determined at different time points (n = 9) by western blotting of brain extracts, using an antibody against BMAL1. Grey bars = dsAST2, white bars = dsGFP. E) Relative amounts of AST2- Pl RACK1- Pl BMAL1 protein complex in the brain in vivo was determined at different time points (n = 9) by western blotting of brain extracts, using an antibody against BMAL1. Grey bars = dsAST2, white bars = dsGFP. F) Relative amounts of AST2- Pl RACK1 protein complex in the brain in vivo was determined at different time points (n = 9) by western blotting of brain extracts, using an antibody against RACK1. Grey bars = dsAST2, white bars = dsGFP. G) Relative amounts of AST2 protein in the brain in vivo was determined at different time points (n = 3) as determined by western blotting of brain extracts, using an antibody against AST2. Grey bars = dsAST2, white bars = dsGFP. Quantity One analysis was used to quantify the intensity of protein bands from three independent experiments and results are presented in graphs D to G. Statistical significance: *P<0.05, **P<0.01 using Student's paired t-test (error bars indicate SD from nine replicates).

Article Snippet: A goat polyclonal antibody to human BMAL1 (N-5: SC-8550) and goat polyclonal antibody to mouse CLK (S-19: SC-6927) were obtained from Santa Cruz Biotechnology.

Techniques: Expressing, Knockdown, Injection, Control, In Vivo, Western Blot

Journal: PLoS Genetics

Article Title: Astakine 2—the Dark Knight Linking Melatonin to Circadian Regulation in Crustaceans

doi: 10.1371/journal.pgen.1003361

Figure Lengend Snippet: A) Crayfish received dsGFP or dsAST2 treatments, then crude brain lysates were harvested during day and night, and were used to study E-box binding with fluorophore labeled oligonucleotides in an EMSA assay. As a control the brain lysates were incubated with an antibody against CLK, to show a supershift. The E-box binding activity of CLK- Pl BMAL1 is indicated on the right side of the gel. B) The effect of AST2 knock down on CLK- Pl BMAL1 E-box binding was analyzed by EMSA. A mutant E-box was used as control. C) Western blot showing the effect of AST2 knock down or melatonin treatment on the protein levels of CLK/ Pl BMAL1, AST2- Pl RACK1- Pl BMAL1 complexes (upper panel), and AST2 level (middle panel). Actin was used as an internal control (lower panel). The horizontal band on top of this histogram indicates day (white) or night (black). D) Relative amounts of CLK- Pl BMAL1 and AST2 -Pl RACK1- Pl BMAL1 protein complexes in brain extracts isolated during the day (n = 9) as determined by western blotting, using an antibody against BMAL1. The AST2 protein was also detected by western blotting, using an antibody against AST2. White bars = dsAST2, black bars = melatonin, grey bars = dsGFP. E) Relative amounts of CLK- Pl BMAL1 and AST2 -Pl RACK1- Pl BMAL1 protein complexes in brain extracts isolated during the night (n = 9) as determined by western blotting, using an antibody against BMAL1. The AST2 protein was also detected by western blotting, using an antibody against AST2. White bars = dsAST2, and grey bars = dsGFP. In three independent experiments, proteins were visualized by western blotting and quantitated using the Quantity One software. These results are shown in graph D and E. Error bars indicate SD from nine replicates. The asterisks indicate significant differences (*P<0.05, **P<0.01); one-way ANOVA with Duncan's new multiple-range test and the Tukey test.

Article Snippet: A goat polyclonal antibody to human BMAL1 (N-5: SC-8550) and goat polyclonal antibody to mouse CLK (S-19: SC-6927) were obtained from Santa Cruz Biotechnology.

Techniques: Binding Assay, Labeling, Control, Incubation, Activity Assay, Knockdown, Mutagenesis, Western Blot, Isolation, Software

Journal: PLoS Genetics

Article Title: Astakine 2—the Dark Knight Linking Melatonin to Circadian Regulation in Crustaceans

doi: 10.1371/journal.pgen.1003361

Figure Lengend Snippet: The protein level of CLK is enhanced during the light period, and a CLK- Pl BMAL1 complex is formed that acts as a transcriptional activator. During the dark period melatonin secretion causes an upregulation of AST2 and this high AST2 level results in the formation of a complex between Pl BMAL1 and Pl RACK1. Then AST2 binds to PlRACK 1 in the PlBMAL-PlRACK1 complex and forms the AST2- Pl RACK1- Pl BMAL1 complex and this interferes with the formation and activity of the CLK- Pl BMAL1 heterodimer.

Article Snippet: A goat polyclonal antibody to human BMAL1 (N-5: SC-8550) and goat polyclonal antibody to mouse CLK (S-19: SC-6927) were obtained from Santa Cruz Biotechnology.

Techniques: Activity Assay

Journal: iScience

Article Title: cGAS and DDX41-STING mediated intrinsic immunity spreads intercellularly to promote neuroinflammation in SOD1 ALS model

doi: 10.1016/j.isci.2022.104404

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-β-tubulin (G8) , Santa Cruz Biotechnology , Catsc-55529; RRID: AB_2210962.

Techniques: Virus, Recombinant, High Molecular Weight, Protease Inhibitor, Blocking Assay, CyQUANT Assay, SYBR Green Assay, ATP Assay, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Luciferase, Control, Software

Journal: Biomolecules

Article Title: Identification of a Novel Antibacterial Function of Mammalian Calreticulin

doi: 10.3390/biom15070966

Figure Lengend Snippet: Evolutionary conservation and sequence homology analysis of calreticulin across multiple species. ( A ) Phylogenetic analysis of calreticulin amino acid sequences from different species. The neighbor-joining phylogenetic tree was constructed using the bootstrap method in MEGA version 10.2.2 with 1000 bootstrap replicates. ( B ) Sequence homology analysis of calreticulin amino acid sequences among various species. The matrix shown represents a bidirectional pairwise comparison of calreticulin sequences. The upper triangular region displays the percentage of sequence identity (% identity), while the lower triangular region shows the corresponding percentage of sequence divergence (% divergence) between each pair of species. Black squares along the diagonal indicate self-alignments, where sequence identity is 100% and divergence is 0%.

Article Snippet: Antibodies: GAPDH antibody (Proteintech, Wuhan, China, 60004-1-Ig, Western blot, 1:1000); calreticulin antibody (Proteintech, 10292-1-AP, Western blot, 1:1000; IHC, 1:200); His-tag (Proteintech, 66005-1-Ig, Western blot, 1:10,000).

Techniques: Sequencing, Construct, Comparison

Journal: Biomolecules

Article Title: Identification of a Novel Antibacterial Function of Mammalian Calreticulin

doi: 10.3390/biom15070966

Figure Lengend Snippet: Distribution of calreticulin in different goat organs. ( A ) Immunohistochemical staining illustrating the localization of calreticulin in the nasal mucosa, pharynx, trachea, lung, heart, liver, spleen, and small intestine of goats. Bars = 50 μm. ( B ) Analysis of calreticulin protein expression levels via Western blot in the nasal mucosa, pharynx, trachea, lung, heart, liver, spleen, and small intestine of goats. ( C ) The band obtained by Western blot was analyzed by gray value, and the result was expressed as the gray value ratios of the CALR/GAPDH. All data shown are the mean ± SD from three independent experiments. Original Western blot images can be found in .

Article Snippet: Antibodies: GAPDH antibody (Proteintech, Wuhan, China, 60004-1-Ig, Western blot, 1:1000); calreticulin antibody (Proteintech, 10292-1-AP, Western blot, 1:1000; IHC, 1:200); His-tag (Proteintech, 66005-1-Ig, Western blot, 1:10,000).

Techniques: Immunohistochemical staining, Staining, Expressing, Western Blot

Journal: Biomolecules

Article Title: Identification of a Novel Antibacterial Function of Mammalian Calreticulin

doi: 10.3390/biom15070966

Figure Lengend Snippet: Expression and purification of recombinant goat calreticulin using the Pichia pastoris expression system. ( A ) Schematic representation of the recombinant plasmid PpIC9K-CALR. ( B ) Agarose gel electrophoresis was performed to analyze the original recombinant plasmid (lane 1), the plasmid digested with EcoRI alone (lane 2), and the plasmid digested with both EcoRI and NotI (lane 3). ( C ) SDS-PAGE analysis of recombinant calreticulin expression in methanol-induced positive transformants, stained with Coomassie Brilliant Blue: Lane 1—culture supernatant of methanol-induced transformants; Lane 2—flow-through during Ni-NTA affinity purification; Lane 3—wash fraction; Lane 4—eluted protein with 200 mM imidazole; Lane 5—desalted and concentrated protein following ultrafiltration. ( D ) Western blot analysis confirming the identity of purified recombinant goat calreticulin (lane order as in panel ( C )). ( E ) SEC-HPLC analysis of the molecular weight and purity of yeast-secreted recombinant goat calreticulin. Original Western blot images can be found in .

Article Snippet: Antibodies: GAPDH antibody (Proteintech, Wuhan, China, 60004-1-Ig, Western blot, 1:1000); calreticulin antibody (Proteintech, 10292-1-AP, Western blot, 1:1000; IHC, 1:200); His-tag (Proteintech, 66005-1-Ig, Western blot, 1:10,000).

Techniques: Expressing, Purification, Recombinant, Plasmid Preparation, Agarose Gel Electrophoresis, SDS Page, Staining, Affinity Purification, Western Blot, Molecular Weight

Journal: Biomolecules

Article Title: Identification of a Novel Antibacterial Function of Mammalian Calreticulin

doi: 10.3390/biom15070966

Figure Lengend Snippet: Antibacterial activity of recombinant goat calreticulin. ( A – C ) CFU assays were performed to evaluate the inhibitory effects of calreticulin against Escherichia coli , Salmonella typhimurium , and Pasteurella multocida . ( D – F ) Growth curve analysis of Escherichia coli , Salmonella typhimurium , and Pasteurella multocida in the presence of calreticulin to assess its impact on bacterial proliferation. All data are presented as mean ± SD from three independent experiments. Statistical significance was determined using one-way ANOVA. ns, no significance; *** p < 0.001.

Article Snippet: Antibodies: GAPDH antibody (Proteintech, Wuhan, China, 60004-1-Ig, Western blot, 1:1000); calreticulin antibody (Proteintech, 10292-1-AP, Western blot, 1:1000; IHC, 1:200); His-tag (Proteintech, 66005-1-Ig, Western blot, 1:10,000).

Techniques: Activity Assay, Recombinant

Journal: Biomolecules

Article Title: Identification of a Novel Antibacterial Function of Mammalian Calreticulin

doi: 10.3390/biom15070966

Figure Lengend Snippet: Recombinant goat calreticulin binds to and agglutinates bacteria. ( A – C ) ELISA analysis of bacterial binding: Escherichia coli , Salmonella typhimurium , and Pasteurella multocida (1 × 10 7 CFU/mL) were immobilized on microtiter plates and incubated with either CaCl 2 (10 mM), His-tag peptide (100 μg/mL), His-tag peptide (100 μg/mL) + CaCl 2 (10 mM), calreticulin (100 μg/mL), or calreticulin (100 μg/mL) + CaCl 2 (10 mM). Binding of calreticulin was detected using anti-His tag antibodies. ( D ) Western blot analysis of bacterial pellets after incubation with calreticulin (100 μg/mL) to confirm binding to Escherichia coli , Salmonella typhimurium , and Pasteurella multocida . ( E ) ELISA analysis of calreticulin binding activity to LPS. ( F ) Log-phase Escherichia coli , Salmonella typhimurium , and Pasteurella multocida were labeled with CFSE and incubated with calreticulin (100 μg/mL) ± 10 mM Ca 2+ at 37 °C for 2 h. Bacterial agglutination was visualized by fluorescence microscopy. Bars = 100 μm. All data are expressed as the mean ± SD from three independent experiments. Statistical significance was assessed using one-way ANOVA. ns, no significance; *** p < 0.001. Original Western blot images can be found in .

Article Snippet: Antibodies: GAPDH antibody (Proteintech, Wuhan, China, 60004-1-Ig, Western blot, 1:1000); calreticulin antibody (Proteintech, 10292-1-AP, Western blot, 1:1000; IHC, 1:200); His-tag (Proteintech, 66005-1-Ig, Western blot, 1:10,000).

Techniques: Recombinant, Bacteria, Enzyme-linked Immunosorbent Assay, Binding Assay, Incubation, Western Blot, Activity Assay, Labeling, Agglutination, Fluorescence, Microscopy

Journal: Biomolecules

Article Title: Identification of a Novel Antibacterial Function of Mammalian Calreticulin

doi: 10.3390/biom15070966

Figure Lengend Snippet: Intranasal infection with Pasteurella multocida upregulates calreticulin expression in the respiratory tissues of the lambs. Thirty-day-old lambs were intranasally challenged with Pasteurella multocida and euthanized 24 h post-infection, after which respiratory tract tissues, including the nasal cavity, trachea, and lung, were collected for further analysis. ( A ) Immunohistochemical staining showing calreticulin expression in the nasal mucosa, trachea, and lungs following infection. Bars = 20 μm. ( B ) Quantitative analysis of immunohistochemical staining based on gray value measurements. ( C ) RT-qPCR analysis of calreticulin mRNA expression in different respiratory tissues after Pasteurella multocida infection. ( D ) Western blot analysis of calreticulin protein levels in infected respiratory tissues. ( E ) Densitometric analysis of Western blot results. All data are presented as mean ± SD from three independent experiments. Statistical significance was evaluated using one-way ANOVA. ** p < 0.01; *** p < 0.001. Original Western blot images can be found in .

Article Snippet: Antibodies: GAPDH antibody (Proteintech, Wuhan, China, 60004-1-Ig, Western blot, 1:1000); calreticulin antibody (Proteintech, 10292-1-AP, Western blot, 1:1000; IHC, 1:200); His-tag (Proteintech, 66005-1-Ig, Western blot, 1:10,000).

Techniques: Infection, Expressing, Immunohistochemical staining, Staining, Quantitative RT-PCR, Western Blot

Journal: Biomolecules

Article Title: Identification of a Novel Antibacterial Function of Mammalian Calreticulin

doi: 10.3390/biom15070966

Figure Lengend Snippet: Intranasal administration of calreticulin alleviates Pasteurella multocida -induced pathological injury and promotes bacterial clearance in the lambs. Thirty-day-old lambs were intranasally infected with Pasteurella multocida ; 6 h post-infection, 1 mL of recombinant calreticulin (2.5 mg/mL) was administered intranasally. Lambs were euthanized and necropsied at 24 h post-infection for sample collection. ( A ) Histopathological evaluation of nasal mucosa ( a – c ), trachea ( d – f ), and lung tissues ( g – i ) using H&E staining. Bars = 20 μm. ( B ) Average injury scores calculated for all lambs ( n = 3 replicates/group). ( C ) In situ hybridization with a Pasteurella multocida -specific fluorescent probe to detect bacterial load in nasal cavity ( a , b ), tracheal ( c , d ), and lung ( e , f ) tissue sections. Bars = 100 μm. ( D ) Quantification of fluorescence intensity derived from in situ hybridization results. All data are presented as mean ± SD from three independent experiments. Statistical significance was assessed using one-way ANOVA. *** p < 0.001.

Article Snippet: Antibodies: GAPDH antibody (Proteintech, Wuhan, China, 60004-1-Ig, Western blot, 1:1000); calreticulin antibody (Proteintech, 10292-1-AP, Western blot, 1:1000; IHC, 1:200); His-tag (Proteintech, 66005-1-Ig, Western blot, 1:10,000).

Techniques: Infection, Recombinant, Staining, In Situ Hybridization, Fluorescence, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: Hypothalamic ER–associated degradation regulates POMC maturation, feeding, and age-associated obesity

doi: 10.1172/JCI96420

Figure Lengend Snippet: ( A ) Representative images of immunohistochemical staining of Hrd1 in the brains of 7-week-old C57BL/6J mice on LFD ( n = 2 each group). Zoomed-in images of ARC and thalamus are shown on the right. 3V, third ventricle. Representative images of negative control IgG are shown in . ( B ) Representative images of Hrd1 staining in the ARC of 7-week-old POMC-eGFP reporter mice after an overnight fast with or without 6-hour refeeding. Quantitation of Hrd1 signals in POMC neurons (green arrows) and non-POMC neurons (white arrows) shown on the right ( n = 2 mice each group, 70 neurons each mouse). ( C ) Representative images of Hrd1 staining in the ARC of 8-week-old Sel1L POMC ;POMC-eGFP and control Sel1L POMC/+ ; POMC-eGFP mice on LFD ( n = 3–4 each group). Green arrows point to POMC neurons; white arrows point to non-POMC neurons. ( D ) Quantitation of Hrd1 level shown in C in POMC and non-POMC neurons in the ARC ( n = 70 and n = 100 neurons per mouse, n = 3–4 mice each). ( E and F ) Growth curve of Sel1L fl/fl ( n = 5), heterozygous Sel1L POMC/+ ( Sel1L fl/+ ;Pomc-Cre , n = 3), and Sel1L POMC mice ( n = 7) on LFD. In E , a green dotted line marks the age at which Sel1L POMC mice became significantly more obese. ( G ) Body weight of 10- and 40-week-old mice on LFD. ( H ) Representative image of 40-week-old mice on LFD. ( I ) Body composition of 10-week-old ( n = 3 each) and 40-week-old ( n = 6–7 each) male mice on LFD. ( J and K ) Representative images of peripheral tissues ( J ) and H&E images of peripheral tissues ( K ) from 40-week-old mice ( n = 3 each group). gWAT, gonadal WAT. ( L and M ) Serum leptin ( L ) and insulin ( M ) levels of 8- and 40-week-old mice of both sexes fed ad libitum LFD ( n = approximately 4–6 each group). Values are shown as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001, 2-way ANOVA.

Article Snippet: Antibodies for immunostaining were as follows: HA (mouse, 1:500; catalog H9658, Sigma-Aldrich), XTP3-B (goat, 1:500; catalog sc-161409, Santa Cruz Biotechnology Inc.) and Hrd1 (rabbit, 1:500), POMC (goat, 1:250; catalog NB100-1533, Novus Biologicals), ACTH (rabbit, 1:100; catalog G-001-06, Phoenix Pharmaceuticals), α-MSH (sheep, 1:2,000; catalog AB5087, Millipore), β-endorphin (rabbit, 1:2,000; catalog H-022-33, Phoenix Pharmaceuticals, provided by Carol Elisa), and GFP (chicken, 1:500; catalog GFP-1020, Aves Labs).

Techniques: Immunohistochemical staining, Staining, Negative Control, Quantitation Assay, Control

Journal: The Journal of Clinical Investigation

Article Title: Hypothalamic ER–associated degradation regulates POMC maturation, feeding, and age-associated obesity

doi: 10.1172/JCI96420

Figure Lengend Snippet: ( A ) Schematic diagram showing specific domains and processing derivatives of POMC recognized by various antibodies. SP, signal peptide; β-endo, β-endorphin. ( B – E ) Representative immunofluorescence images of ( B ) POMC as a prohormone ( n = 5–6 each) in the ARC of mice approximately 5 to 10 weeks of age, ( C ) α-MSH in the axons of POMC neurons in the PVN of mice approximately 5 to 10 weeks of age ( n = 4 each), ( D ) β-endorphin ( n = 4–5 in each), and ( E ) ACTH ( n = 2-3 each) in the ARC and DMH of mice approximately 5 to 8 weeks of age fed ad libitum LFD. White arrows indicate staining in the cell bodies of POMC neurons. Quantitation of POMC and neuropeptide signal intensity in cell bodies and axons are shown in . ( F ) Pomc mRNA level in the ARC of mice approximately 5 to 8 weeks of age fed ad libitum LFD ( n = 4 each). ( G ) α-MSH levels in the hypothalamus of mice approximately 6 to 7 weeks of age, measured by ELISA ( n = 3 each group). ( H ) Cumulative food intake in 8-week-old mice injected daily i.p. with α-MSH (1 mg/kg body weight) for 3 days ( n = approximately 5–7 each group). Vehicle, saline. Values are shown as mean ± SEM. * P < 0.05, Student’s t test ( F and G ) or 2-way ANOVA ( H ).

Article Snippet: Antibodies for immunostaining were as follows: HA (mouse, 1:500; catalog H9658, Sigma-Aldrich), XTP3-B (goat, 1:500; catalog sc-161409, Santa Cruz Biotechnology Inc.) and Hrd1 (rabbit, 1:500), POMC (goat, 1:250; catalog NB100-1533, Novus Biologicals), ACTH (rabbit, 1:100; catalog G-001-06, Phoenix Pharmaceuticals), α-MSH (sheep, 1:2,000; catalog AB5087, Millipore), β-endorphin (rabbit, 1:2,000; catalog H-022-33, Phoenix Pharmaceuticals, provided by Carol Elisa), and GFP (chicken, 1:500; catalog GFP-1020, Aves Labs).

Techniques: Immunofluorescence, Staining, Quantitation Assay, Enzyme-linked Immunosorbent Assay, Injection, Saline

Journal: The Journal of Clinical Investigation

Article Title: Hypothalamic ER–associated degradation regulates POMC maturation, feeding, and age-associated obesity

doi: 10.1172/JCI96420

Figure Lengend Snippet: ( A and B ) Steady-state protein levels of POMC in WT and ERAD-deficient N2a cells transfected with POMC-WT-Flag. In B , proteasomal inhibitor MG132 was added for the last 2 hours. mRNA levels of Pomc in A and B are shown in . ( C and D ) Ubiquitination of POMC by HRD1 in gain- ( C ) and loss-of-function ( D ) systems: Western blot of ubiquitin in POMC-Flag immunoprecipitates of HEK293T cells transfected with POMC-WT-Flag with or without HRD1. MG132 ( C ) or bortezomib ( D ) was added for the last 2 hours. In D , quantitation of the ratio of ubiquitination (Ub) signal intensity to POMC band intensity is shown in the lower panels. ( E ) Western blot analysis of POMC-Flag immunoprecipitates in transfected WT and Hrd1 –/– N2a cells under nonreducing (-β-ME) or reducing (+β-ME) SDS-PAGE. ( F ) Western blot analysis of endogenous POMC processing using ACTH antibody in POMC-expressing mouse pituitary tumor line AtT20 with or without Sel1L. ( G ) Representative confocal images of POMC in POMC-transfected WT and Sel1L –/– N2a cells. White arrows point to POMC-containing secretory granules, while yellow arrows point to perinuclear POMC. KDEL marks the ER. Representative data from at least 2 independent experiments are shown.

Article Snippet: Antibodies for immunostaining were as follows: HA (mouse, 1:500; catalog H9658, Sigma-Aldrich), XTP3-B (goat, 1:500; catalog sc-161409, Santa Cruz Biotechnology Inc.) and Hrd1 (rabbit, 1:500), POMC (goat, 1:250; catalog NB100-1533, Novus Biologicals), ACTH (rabbit, 1:100; catalog G-001-06, Phoenix Pharmaceuticals), α-MSH (sheep, 1:2,000; catalog AB5087, Millipore), β-endorphin (rabbit, 1:2,000; catalog H-022-33, Phoenix Pharmaceuticals, provided by Carol Elisa), and GFP (chicken, 1:500; catalog GFP-1020, Aves Labs).

Techniques: Transfection, Ubiquitin Proteomics, Western Blot, Quantitation Assay, SDS Page, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Hypothalamic ER–associated degradation regulates POMC maturation, feeding, and age-associated obesity

doi: 10.1172/JCI96420

Figure Lengend Snippet: ( A ) Schematic diagram showing amino acid sequence of POMC 26–50 and the positions of 2 disulfide bonds and a free thiol in POMC- C28F . ( B ) Western blot analysis of steady-state levels of POMC proteins in WT and Hrd1 –/– N2a cells transfected with POMC-WT and - C28F . mRNA levels of each sample are shown below. ( C ) Western blot analyses of ubiquitination following immunoprecipitation of POMC in HEK293T cells transfected with POMC-WT-Flag or POMC- C28F -Flag construct with or without HA-Ub, Myc-tagged HRD1-WT, or HRD1 E3 ligase-dead C2A mutant. ( D ) Representative confocal images of POMC in POMC-transfected WT and Sel1L –/– N2a cells. White arrows point to secreted POMC in granules, while yellow arrows point to perinuclear POMC, possibly in the form of aggregates. KDEL marks the ER. ( E ) Sucrose gradient fractionation (fractions 1–7 from top to bottom of gradient) of HEK293T cells expressing POMC-WT or - C28F under nonreducing (–β-ME) and reducing (+β-ME) SDS-PAGE. ( F ) Western blot analysis of Myc-tagged POMC in WT and Hrd1 –/– N2a cells transfected with POMC-WT or - C28F under reducing and nonreducing SDS-PAGE. Red box marks HMW aggregates, while green box marks monomers and dimers. HMW, high molecular weight. (POMC) 2 , POMC dimers. Representative data from at least 2 independent experiments are shown.

Article Snippet: Antibodies for immunostaining were as follows: HA (mouse, 1:500; catalog H9658, Sigma-Aldrich), XTP3-B (goat, 1:500; catalog sc-161409, Santa Cruz Biotechnology Inc.) and Hrd1 (rabbit, 1:500), POMC (goat, 1:250; catalog NB100-1533, Novus Biologicals), ACTH (rabbit, 1:100; catalog G-001-06, Phoenix Pharmaceuticals), α-MSH (sheep, 1:2,000; catalog AB5087, Millipore), β-endorphin (rabbit, 1:2,000; catalog H-022-33, Phoenix Pharmaceuticals, provided by Carol Elisa), and GFP (chicken, 1:500; catalog GFP-1020, Aves Labs).

Techniques: Sequencing, Western Blot, Transfection, Ubiquitin Proteomics, Immunoprecipitation, Construct, Mutagenesis, Fractionation, Expressing, SDS Page, High Molecular Weight

Journal: The Journal of Clinical Investigation

Article Title: Hypothalamic ER–associated degradation regulates POMC maturation, feeding, and age-associated obesity

doi: 10.1172/JCI96420

Figure Lengend Snippet: ( A ) Under physiological conditions, Sel1L-Hrd1 ERAD plays an important role in promoting a conducive environment for the maturation of nascent POMC in the ER by degrading, likely misfolded, POMC. ( B ) In the absence of ERAD, misfolded POMC accumulates and interferes with the maturation of nascent POMC in the ER in a disulfide bond–dependent manner. ( C ) Under pathological conditions, POMC- C28F is consistently misfolded and forms aggregates via intermolecular C50-mediated disulfide bonds. This model explains the dominant-negative effect of POMC- C28F . ( D ) The maturation defects of POMC- C28F can be completely rescued by an intragenic suppressor mutation, C50S .

Article Snippet: Antibodies for immunostaining were as follows: HA (mouse, 1:500; catalog H9658, Sigma-Aldrich), XTP3-B (goat, 1:500; catalog sc-161409, Santa Cruz Biotechnology Inc.) and Hrd1 (rabbit, 1:500), POMC (goat, 1:250; catalog NB100-1533, Novus Biologicals), ACTH (rabbit, 1:100; catalog G-001-06, Phoenix Pharmaceuticals), α-MSH (sheep, 1:2,000; catalog AB5087, Millipore), β-endorphin (rabbit, 1:2,000; catalog H-022-33, Phoenix Pharmaceuticals, provided by Carol Elisa), and GFP (chicken, 1:500; catalog GFP-1020, Aves Labs).

Techniques: Dominant Negative Mutation, Mutagenesis

Journal: Peritoneal Dialysis International : Journal of the International Society for Peritoneal Dialysis

Article Title: Glucose-Based Peritoneal Dialysis Solution Suppresses Adiponectin Synthesis Through Oxidative Stress in an Experimental Model of Peritoneal Dialysis

doi: 10.3747/pdi.2009.00228

Figure Lengend Snippet: — Effect of peritoneal dialysis solution (PDS) with and without N-acetylcysteine (NAC) on drain volume (A), plasma glucose (B), and adiponectin levels in plasma (C) and spent dialysate (D) in rat. After 12 weeks of PD, a peritoneal equilibrium test using 3.86% glucose PDS was performed immediately before the rats were sacrificed; plasma and spent dialysate were collected and analyzed. Values are mean ± SE of 8 animals. *p < 0.05 versus sham, †p < 0.05 versus PDS-treated rats.

Article Snippet: A commercial ELISA kit for rat adiponectin (R&D Systems, Minneapolis, MN, USA) was used to quantify adiponectin in plasma and spent dialysate and a kit for mouse adiponectin (R&D Systems) was used for 3T3-L1 adipocyte culture media according to manufacturers’ instructions.

Techniques: Clinical Proteomics

Journal: Peritoneal Dialysis International : Journal of the International Society for Peritoneal Dialysis

Article Title: Glucose-Based Peritoneal Dialysis Solution Suppresses Adiponectin Synthesis Through Oxidative Stress in an Experimental Model of Peritoneal Dialysis

doi: 10.3747/pdi.2009.00228

Figure Lengend Snippet: — Differentiation of 3T3-L1 preadipocytes. Images of mouse cells were captured on days 0, 4, and 10 by light microscope. Inset pictures represent Oil Red O staining. 3T3-L1 cells were fully differentiated at day 10 (A; right panel). Adiponectin mRNA expression level during 3T3-L1 cell differentiation was examined by RT-PCR (B). Adiponectin secretion in the media was measured by ELISA after 24-hour incubation under indicated experimental condition (C). Relative secretion was calculated as the ratio of adiponectin to protein (μg/mg). Basal adiponectin secreted in the media was 6.0 ± 0.7 μg/mg protein. Values are mean ± SE of 4 – 5 independent experiments. *p < 0.05 versus control; †p < 0.05 versus H2O2 200 μmol/L. HG = high D-glucose; PDS = peritoneal dialysis solution diluted twofold with DMEM; RT-PCR = reverse-transcription polymerase chain reaction.

Article Snippet: A commercial ELISA kit for rat adiponectin (R&D Systems, Minneapolis, MN, USA) was used to quantify adiponectin in plasma and spent dialysate and a kit for mouse adiponectin (R&D Systems) was used for 3T3-L1 adipocyte culture media according to manufacturers’ instructions.

Techniques: Light Microscopy, Staining, Expressing, Cell Differentiation, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Incubation, Control, Reverse Transcription, Polymerase Chain Reaction

Journal: Peritoneal Dialysis International : Journal of the International Society for Peritoneal Dialysis

Article Title: Glucose-Based Peritoneal Dialysis Solution Suppresses Adiponectin Synthesis Through Oxidative Stress in an Experimental Model of Peritoneal Dialysis

doi: 10.3747/pdi.2009.00228

Figure Lengend Snippet: — Effect of H2O2 on adiponectin oligomer secretion (A), adiponectin mRNA expression during 3T3-L1 cell differentiation (B), and in adipocytes freshly isolated from mouse abdominal fat pads (C). The effect of H2O2 (H; 200 μmol/L) on multimer, hexamer, and trimer adiponectin secretion was evaluated under nonreducing conditions. mRNA expression levels were examined by RT-PCR and real-time PCR. Values are mean ± SE of 3 – 4 independent experiments. *p < 0.05 versus control (C) at each time point. RT-PCR = reverse-transcription polymerase chain reaction; HMW = high-molecular weight 12- to 18-mer; MMW = middle-molecular weight hexamer; LMW= low-molecular weight trimer.

Article Snippet: A commercial ELISA kit for rat adiponectin (R&D Systems, Minneapolis, MN, USA) was used to quantify adiponectin in plasma and spent dialysate and a kit for mouse adiponectin (R&D Systems) was used for 3T3-L1 adipocyte culture media according to manufacturers’ instructions.

Techniques: Expressing, Cell Differentiation, Isolation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Control, Reverse Transcription, Polymerase Chain Reaction, High Molecular Weight, Molecular Weight