mouse monoclonal anti fgf21 antibody  (R&D Systems)

Journal: Cell reports

Article Title: miRNA-32 Drives Brown Fat Thermogenesis and Trans-activates Subcutaneous White Fat Browning in Mice

doi: 10.1016/j.celrep.2017.04.035

Figure Lengend Snippet: (A) Cold-induced increase in serum FGF21 levels was blunted by injection of miR-32-ASO as measured by ELISA (n = 7). (B) qRT-PCR showed that cold stress decreased FGF21 mRNA expression in liver but increased it in BAT. Injection of miR-32-ASO significantly repressed the increase in FGF21 expression in BAT during cold exposure when normalized to PPIA . (C) FGF21 immunostaining showed that cold stress decreased FGF21 level in liver but increased it in BAT. Injection of miR-32-ASO significantly repressed the increase in FGF21 expression in BAT during cold exposure. (D) Quantification of FGF21 immunostaining in BAT showed that FGF21 levels within BAT increased greatly after cold exposure but were significantly lower in miR-32-ASO-treated mice compared to control-ASO-treated mice. (E) Quantification of FGF21 immunostaining in liver showed that FGF21 levels within liver decreased greatly after cold exposure but were similar between miR-32-ASO-treated mice and control-ASO-treated mice. (F) Western blot showing cold-induced increases in BAT FGF21 protein levels were blunted by injection of miR-32-ASO. Calnexin served as a loading control. (G) Western blot showing cold-induced decreases in liver FGF21 protein levels were unaffected by injection of miR-32-ASO. Calnexin served as a loading control. (H) Quantification of the western blot results in (F) and (G) using ImageJ. Average intensities were normalized to that of Calnexin. (I) FGF21 mRNA expression in WT-1 cells increased or decreased when transfected with miR-32 mimic or miR-32-ASO, respectively. (J) Western blotting showed that FGF21 protein levels in WT-1 cells increased or decreased when transfected with miR-32 mimic or miR-32-ASO, respectively. Data represent mean ± SEM. *p < 0.05 and ** p < 0.01. See also .

Article Snippet: Thermo Scientific 96-well, clear, flat-bottom plates (Nunc) were coated overnight at 4°C with a mouse monoclonal anti-FGF21 antibody (MAB25371, R&D Systems) at 1:1,500 dilution in Na 2 CO 3 /NaHCO 3 buffer at pH 9.6.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Immunostaining, Control, Western Blot, Transfection

Journal: Cell reports

Article Title: miRNA-32 Drives Brown Fat Thermogenesis and Trans-activates Subcutaneous White Fat Browning in Mice

doi: 10.1016/j.celrep.2017.04.035

Figure Lengend Snippet: (A) High mirSVR score and favorable binding energy from bioinformatic analysis predicted that miR-32 directly targets the 3′ UTR of Tob1 . (B) The complementary sequence in the Tob1 3′ UTR and the seed region of miR-32 (red letters) are conserved among mammalian species. (C) Tob1 mRNA level in WT-1 cells decreased or increased 24 hr after transfection of miR-32 mimic or miR-32-ASO, respectively. (D) Tob1 protein level decreased or increased in WT-1 cells when transfected with miR-32 mimic or miR-32-ASO, respectively. (E and F) Activities of a luciferase reporter gene linked to the Tob1 3′ UTR decreased or increased 24 hr after transfection of miR-32 mimic (E) or miR-32-ASO (F), respectively. (G and H) Site-directed mutagenesis of the Tob1 3′ UTR sequence complimentary to the miR-32 seed sequence abolished the effects of miR-32 mimic (G) and miR-32-ASO (H) transfection on the luciferase activity. (I) Knockdown of Tob1 by siTob1 suppressed the increase in Tob1 expression and rescued the decrease in FGF21 expression caused by miR-32-ASO transfection. (J) Western blots showed that siTob1 suppressed the increase in Tob1 expression and rescued the decreases of phospho-p38, phospho-ATF2, and FGF21 expression caused by miR-32-ASO transfection. Numbers indicate protein levels quantified using ImageJ relative to control-ASO. (K) qRT-PCR showed miR-32-ASO-treated mice failed to fully downregulate Tob1 level in response to cold stimulation. (L) Western blots showed miR-32-ASO-treated mice fail to downregulate Tob1 levels and activate p38 and ATF2 phosphorylation in BAT in response to cold stimulation. (M) Quantification of relative protein expression in BAT from the western blots showed in (L) using ImageJ. Average intensities were normalized to that of Calnexin. (N) Cold-exposure time course showing miR-32 and Tob1 expression in BAT at various time points after cold exposure. (O) Cold-exposure time course showing FGF21 expression in BAT at various time points after cold exposure. (P) Cold-exposure time course showing Tob1, UCP1, and FGF21 protein expression in BAT at various time points after cold exposure. (Q) Cold-exposure time course showing UCP1 protein expression in iWAT at various time points after cold exposure. Data represent mean ± SEM. *p < 0.05 and ** p < 0.01. See also .

Article Snippet: Thermo Scientific 96-well, clear, flat-bottom plates (Nunc) were coated overnight at 4°C with a mouse monoclonal anti-FGF21 antibody (MAB25371, R&D Systems) at 1:1,500 dilution in Na 2 CO 3 /NaHCO 3 buffer at pH 9.6.

Techniques: Binding Assay, Sequencing, Transfection, Luciferase, Mutagenesis, Activity Assay, Knockdown, Expressing, Western Blot, Control, Quantitative RT-PCR, Phospho-proteomics

Journal: Cell reports

Article Title: miRNA-32 Drives Brown Fat Thermogenesis and Trans-activates Subcutaneous White Fat Browning in Mice

doi: 10.1016/j.celrep.2017.04.035

Figure Lengend Snippet: (A) miR-32 expression in BAT but not in liver was blunted in BAT-specific miR-32-ASO-injected mice (n = 8) compared to control-ASO-injected mice (n = 7). (B) miR-32-ASO-BS mice (n = 8) showed lower core body temperatures during cold exposure when compared to control-ASO-BS mice (n = 7). (C) Total energy expenditure was significantly reduced after 7 days’ cold stress in miR-32-ASO-BS mice (n = 6) as compared with control-ASO-BS mice (n = 6). Energy expenditure was normalized to lean body mass. (D) Average total energy expenditure was significantly lower in miR-32-ASO-BS mice (n = 6). (E) Percentage BAT mass was significantly lower in miR-32-ASO-BS mice (n = 8) compared to control mice (n = 7). (F) In BAT, mRNA levels of Tob1 were higher in miR-32-ASO-BS mice (n = 8), whereas expression of thermogenic genes was significantly lower in miR-32-ASO-BS mice. (G) Protein levels of Tob1 were higher in miR-32-ASO-BS mice (n = 8) compared to control mice (n = 7). However, p-p38, p-ATF2, FGF21, UCP1, and PGC1α were lower in miR-32-ASO-BS mice (n = 8). Total p38 and total ATF2 expression were not significantly different between the two groups. (H) Quantification of relative protein expression using ImageJ showed that protein level of Tob1 was upregulated, but p-p38, p-ATF2, FGF21, UCP1, and PGC1α were significantly lower in BAT of miR-32-ASO-BS mice (n = 8) compared to control mice (n = 7). Total p38 and total ATF2 expression were comparable between the two groups. Average intensities were normalized to that of Calnexin. (I) Serum FGF21 levels were decreased in miR-32-ASO-BS mice (n = 8) compared to control mice (n = 7). (J) mRNA levels of thermogenic genes in iWAT were significantly reduced in miR-32-ASO-BS mice. Data were normalized to PPIA . (K) Immunoblots showed that miR-32-ASO-BS mice (n = 8) had significantly reduced UCP1 and PGC1α protein levels in iWAT compared to control-ASO-BS mice (n = 7). (L) Quantification of relative UCP1 and PGC1α protein levels using ImageJ. Average intensities were normalized to that of Calnexin. Data represent mean ± SEM. *p < 0.05, **p < 0.01, and *** p < 0.001. See also .

Article Snippet: Thermo Scientific 96-well, clear, flat-bottom plates (Nunc) were coated overnight at 4°C with a mouse monoclonal anti-FGF21 antibody (MAB25371, R&D Systems) at 1:1,500 dilution in Na 2 CO 3 /NaHCO 3 buffer at pH 9.6.

Techniques: Expressing, Injection, Control, Western Blot

Journal: Cell reports

Article Title: miRNA-32 Drives Brown Fat Thermogenesis and Trans-activates Subcutaneous White Fat Browning in Mice

doi: 10.1016/j.celrep.2017.04.035

Figure Lengend Snippet: (A) miR-32 expression in BAT but not in liver or iWAT was increased in miR-32-AAV-BS mice (n = 8) compared to control-AAV-BS mice (n = 7). (B) miR-32-AAV-BS mice (n = 8) showed higher core body temperatures during cold exposure when compared to control-AAV-BS mice (n = 7). (C) Total energy expenditure was significantly increased after 7 days’ cold stress in miR-32-AAV-BS mice (n = 6) as compared with control-AAV-BS mice (n = 6). Energy expenditure was normalized to lean body mass. (D) Average total energy expenditure was significantly higher in miR-32-AAV-BS mice (n = 6). (E) Percentage BAT mass was significantly higher in miR-32-AAV-BS mice (n = 8) compared to control-AAV-BS mice (n = 7). (F) In BAT, mRNA levels of Tob1 were lower in miR-32-AAV-BS mice, whereas expression of thermogenic genes was significantly higher in miR-32-AAV-BS mice. (G) Protein levels of Tob1 were lower in miR-32-AAV-BS mice (n = 8) compared to control-AAV-BS mice (n = 7). However, p-p38, p-ATF2, FGF21, UCP1, and PGC1α were higher in miR-32-AAV-BS mice (n = 8). Total p38 and total ATF2 expression were not significantly different between the two groups. (H) Quantification of relative protein expression using ImageJ showed that protein level of Tob1 was reduced, but p-p38, p-ATF2, FGF21, UCP1, and PGC1α were significantly higher in BAT of miR-32-AAV-BS mice compared to control. Total p38 and total ATF2 expression were comparable between the two groups. Average intensities were normalized to that of Calnexin. (I) Serum FGF21 levels were increased in miR-32-AAV-BS mice (n = 8) compared to control-AAV-BS mice (n = 7). (J) mRNA levels of thermogenic genes in iWAT were significantly increased in miR-32-AAV-BS mice. Data were normalized to PPIA . (K) Immunoblots showed that miR-32-AAV-BS mice (n = 8) had significantly increased UCP1 and PGC1α protein levels in iWAT compared to control-AAV-BS mice (n = 7). (L) Quantification of relative UCP1 and PGC1α protein levels using ImageJ. Average intensities were normalized to that of Calnexin. Data represent mean ± SEM. *p < 0.05, **p < 0.01, and *** p < 0.001. See also .

Article Snippet: Thermo Scientific 96-well, clear, flat-bottom plates (Nunc) were coated overnight at 4°C with a mouse monoclonal anti-FGF21 antibody (MAB25371, R&D Systems) at 1:1,500 dilution in Na 2 CO 3 /NaHCO 3 buffer at pH 9.6.

Techniques: Expressing, Control, Western Blot

Journal: Cell reports

Article Title: miRNA-32 Drives Brown Fat Thermogenesis and Trans-activates Subcutaneous White Fat Browning in Mice

doi: 10.1016/j.celrep.2017.04.035

Figure Lengend Snippet: (A) FGF21 mRNA expression in BAT but not in liver or iWAT was ablated in miR-32-AAV-BS+Cre mice (n = 6) and control-AAV-BS+Cre mice (n = 6). (B) FGF21 protein expression in BAT but not in liver or iWAT was ablated in miR-32-AAV-BS+Cre mice (n = 6) and control-AAV-BS+Cre mice (n = 6). (C) Quantification of relative protein expression using ImageJ showed that protein level of FGF21 in BAT but not in liver or iWAT was ablated in miR-32-AAV-BS+Cre mice (n = 6) and control-AAV-BS+Cre mice (n = 6). (D) Serum FGF21 levels were decreased in miR-32-AAV-BS+Cre mice (n = 6) and control-AAV-BS+Cre mice (n = 6) compared to wild-type mice (n = 4). (E) miR-32-AAV-BS+Cre mice (n = 6) showed higher core body temperatures only during first 48 hr of cold exposure when compared to control-AAV-BS+Cre mice (n = 6). (F) Total energy expenditure was similar after 7 days’ cold stress in miR-32-AAV-BS+Cre mice (n = 6) as compared with control-AAV-BS+Cre mice (n = 6). Energy expenditure was normalized to lean body mass. (G) Average total energy expenditure was slightly higher in miR-32-AAV-BS+Cre mice (n = 6) than control-AAV-BS+Cre mice (n = 6) but not statistically significant. (H) Percentage BAT mass was slightly higher in miR-32-AAV-BS+Cre mice (n = 6) compared to control-AAV-BS+Cre mice (n = 6). (I) In BAT, mRNA levels of Tob1 were lower in miR-32-AAV-BS+Cre mice (n = 6) compared to control-AAV-BS+Cre mice (n = 6), whereas expression of several thermogenic genes including UCP1 was higher in miR-32-AAV-BS+Cre mice. (J) Protein levels of PGC1α and UCP1 were higher in miR-32-AAV-BS+Cre mice (n = 6) compared to control-AAV-BS+Cre mice (n = 6). (K) Quantification of relative protein expression using ImageJ showed that protein levels of PGC1 and UCP1 were higher in miR-32-AAV-BS+Cre mice (n = 6) compared to control-AAV-BS+Cre mice (n = 6). Average intensities were normalized to that of Calnexin. (L) mRNA levels of thermogenic genes in iWAT were similar in both groups of mice (both n = 6). Data were normalized to PPIA . (M) Immunoblots showed that miR-32-AAV-BS+Cre mice (n = 6) had similar UCP1 and PGC1α protein levels in iWAT compared to control-AAV-BS+Cre mice (n = 6). (N) Quantification of relative UCP1 and PGC1α protein levels using ImageJ. Average intensities were normalized to that of Calnexin. (O) Proposed mechanism by which miR-32 promotes BAT thermogenesis by inhibiting Tob1, activating p38/MAPK signaling and driving UCP1, PGC1α, and FGF21 expression in BAT. The BAT secreted FGF21 functions in a paracrine fashion to promote further thermogenic gene expression in BAT as well as in an endocrine fashion to promote iWAT browning. Data represent mean ± SEM. *p < 0.05, **p < 0.01, and *** p < 0.001. See also .

Article Snippet: Thermo Scientific 96-well, clear, flat-bottom plates (Nunc) were coated overnight at 4°C with a mouse monoclonal anti-FGF21 antibody (MAB25371, R&D Systems) at 1:1,500 dilution in Na 2 CO 3 /NaHCO 3 buffer at pH 9.6.

Techniques: Expressing, Control, Western Blot, Gene Expression

Journal: Molecular medicine reports

Article Title: Expression and purification of FGF21 in Pichia pastoris and its effect on fibroblast-cell migration.

doi: 10.3892/mmr.2016.4942

Figure Lengend Snippet: Figure 2. Expression patterns of FGF21 and β‑klotho in skin following wounding. (A) Reverse‑transcription polymerase chain reaction analysis was used to analyze the expression of FGF21 and β‑klotho prior to and 3 h after wounding. Expression levels of (B) FGF21 and (C) β‑klotho were normalized against those of GAPDH. The experiments were repeated at least three times and values are expressed as the mean ± standard error of three replicates. ***P<0.001 vs. Con. Con, control.

Article Snippet: Subsequently, cells were cultured for 72 h with addition of 0.8% methanol every 24 h. Proteins were separated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Beyotime Institute of Biotechnology, Shanghai, China) and western blot analysis was performed using mouse anti-FGF21 monoclonal antibody (1:1,000; MAB25371; R&D Systems, Inc., Minneapolis, MN, USA) (18).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Molecular medicine reports

Article Title: Expression and purification of FGF21 in Pichia pastoris and its effect on fibroblast-cell migration.

doi: 10.3892/mmr.2016.4942

Figure Lengend Snippet: Figure 1. FGF21 expression patterns in a variety of mouse tissues. (A) Expression patterns of FGF21 were analyzed by reverse‑transcription polymerase chain reaction analysis. (B) FGF21 levels in A were normal ized against those of GAPDH. The experiments were repeated at least three times and values are expressed as the mean ± standard deviation of three replicates. **P<0.01; ***P<0.001 vs. heart FGF21 levels.

Article Snippet: Subsequently, cells were cultured for 72 h with addition of 0.8% methanol every 24 h. Proteins were separated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Beyotime Institute of Biotechnology, Shanghai, China) and western blot analysis was performed using mouse anti-FGF21 monoclonal antibody (1:1,000; MAB25371; R&D Systems, Inc., Minneapolis, MN, USA) (18).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

Journal: Molecular medicine reports

Article Title: Expression and purification of FGF21 in Pichia pastoris and its effect on fibroblast-cell migration.

doi: 10.3892/mmr.2016.4942

Figure Lengend Snippet: Figure 4. Polymerase chain reaction analysis confirmed that the FGF21 frag ment was successfully integrated into SMD1168 colonies. Lanes 1‑10 are colonies selected from a minimal dextrose plate. Yeast cells transfected with empty vector was used as a negative control. M, marker.

Article Snippet: Subsequently, cells were cultured for 72 h with addition of 0.8% methanol every 24 h. Proteins were separated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Beyotime Institute of Biotechnology, Shanghai, China) and western blot analysis was performed using mouse anti-FGF21 monoclonal antibody (1:1,000; MAB25371; R&D Systems, Inc., Minneapolis, MN, USA) (18).

Techniques: Polymerase Chain Reaction, Transfection, Plasmid Preparation, Negative Control, Marker

Journal: Molecular medicine reports

Article Title: Expression and purification of FGF21 in Pichia pastoris and its effect on fibroblast-cell migration.

doi: 10.3892/mmr.2016.4942

Figure Lengend Snippet: Figure 3. Gene synthesis of FGF21. PCR products of the gene FGF21. Lanes: M, 2000 DNA marker; 1, PCR reaction with primer pair P1/RP1; 2, PCR reaction with primer pair P2/RP2 using the PCR product from the previous cycle as a template; 3‑7, PCR reactions with primer pairs P3/RP3‑P7/RP7, respectively, using the PCR product from the previous round as a template. PCR, polymerase chain reaction.

Article Snippet: Subsequently, cells were cultured for 72 h with addition of 0.8% methanol every 24 h. Proteins were separated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Beyotime Institute of Biotechnology, Shanghai, China) and western blot analysis was performed using mouse anti-FGF21 monoclonal antibody (1:1,000; MAB25371; R&D Systems, Inc., Minneapolis, MN, USA) (18).

Techniques: Marker, Polymerase Chain Reaction

Journal: Molecular medicine reports

Article Title: Expression and purification of FGF21 in Pichia pastoris and its effect on fibroblast-cell migration.

doi: 10.3892/mmr.2016.4942

Figure Lengend Snippet: Figure 6. Effects of exogenous FGF21 treatment on the activation of cell migration and JNK phosphorylation levels. (A) Wound healing assay was performed to analyze the effects of FGF21 (100 ng/ml) in human fibroblasts under low‑glucose (5.5 mM) conditions. Scale bar, 0.05 µm. Magnification, 40x. (B) The cell migra tion distance was quantified from A. (C and D) Following 6 h culture, 100 ng/ml FGF21 was added to the culture medium and gently shaken. Phosphorylation levels of JNK were analyzed after 30 min of FGF21 stimulation. All experiments were performed after incubation with 5 µg/ml mitomycin‑C, an inhibitor of cell proliferation, for one day. Images were captured using an ImageQuant LAS 4000. Values are expressed as the mean ± standard error (n=10 for B and n=3 for D). *P<0.05 vs. control. p‑JNK, phosphorylated c‑Jun N‑terminal kinase; t‑JNK, total c‑Jun N‑terminal kinase; FGF, fibroblast growth factor; Con, control.

Article Snippet: Subsequently, cells were cultured for 72 h with addition of 0.8% methanol every 24 h. Proteins were separated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Beyotime Institute of Biotechnology, Shanghai, China) and western blot analysis was performed using mouse anti-FGF21 monoclonal antibody (1:1,000; MAB25371; R&D Systems, Inc., Minneapolis, MN, USA) (18).

Techniques: Activation Assay, Migration, Phospho-proteomics, Wound Healing Assay, Incubation, Control

Journal: Molecular medicine reports

Article Title: Expression and purification of FGF21 in Pichia pastoris and its effect on fibroblast-cell migration.

doi: 10.3892/mmr.2016.4942

Figure Lengend Snippet: Figure 5. Protein expression and purification of FGF21. (A) SDS‑PAGE analysis of supernatant of engineered P. pastoris cells and purified FGF21 protein. Lanes: 1, marker; 2, supernatant without transfection with the FGF21 overexpression vector; 3, supernatant after transfection; 4, purified protein. (B) Purified FGF21 protein was analyzed by western blotting using an Epson Perfection V700 photo scanner. Lanes: 1, protein in the supernatant after transfection; 2, purified protein. (C) The purity of recombinant FGF21 was analyzed by high‑performance liquid chromatography. FGF, fibroblast growth factor.

Article Snippet: Subsequently, cells were cultured for 72 h with addition of 0.8% methanol every 24 h. Proteins were separated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Beyotime Institute of Biotechnology, Shanghai, China) and western blot analysis was performed using mouse anti-FGF21 monoclonal antibody (1:1,000; MAB25371; R&D Systems, Inc., Minneapolis, MN, USA) (18).

Techniques: Expressing, Purification, Marker, Transfection, Over Expression, Plasmid Preparation, Western Blot, Recombinant, High Performance Liquid Chromatography