protein 4 rbp4 concentrations  (R&D Systems)

Journal: Theranostics

Article Title: Growth hormone receptor disrupts glucose homeostasis via promoting and stabilizing retinol binding protein 4.

doi: 10.7150/thno.61192

Figure Lengend Snippet: Figure 5. Hepatic GHR overexpression results in elevation of serum RBP4 levels. (A) The mRNA levels of hepatokines were determined by qRT-PCR in the livers of AAV-infected mice, which were plotted in a heatmap (n=5). (B) Relative mRNA levels of RBP4 in the livers of AAV-infected mice (n=10). (C) The concentrations of serum RBP4 of AAV-infected mice (n=10). (D) Western blot and SDS-PAGE analysis were performed in the serum of AAV-infected mice. (E) Elution profile of chylomicrons in the serum of AAV-GFP (left) or AAV-GHR (right) mice. Purified proteins were detected in column eluents by monitoring absorbance at 280 nm. (F) Western blots of RBP4 in serum of AAV-GFP (up) or AAV-GHR (down) mice, which were separated by gel filtration chromatography and collected according to the ultraviolet absorption peak of fractions. (G) The correlation between serum RBP4 concentrations and hepatic GHR levels in humans (n=13). Data are expressed as the mean ± SD. ***p < 0.001 (Student’s t-test).

Article Snippet: Moreover, the serum RBP4 concentrations were respectively carried out on humans (#DRB400) and mice (#DRBP40) using corresponding RBP4 Quantikine ELISA Kit (R&D Systems, USA).

Techniques: Over Expression, Quantitative RT-PCR, Infection, Western Blot, SDS Page, Purification, Filtration, Chromatography

Journal: Theranostics

Article Title: Growth hormone receptor disrupts glucose homeostasis via promoting and stabilizing retinol binding protein 4.

doi: 10.7150/thno.61192

Figure Lengend Snippet: Figure 6. GHR activates RBP4 transcription by activating STAT5. (A) Western blots analysis of GHR, p-SAT5 and STAT5 in the livers of AAV-infected mice. (B) The schematic representation of STAT5 binding site in the promoter region of RBP4. (C) Western blots analysis of GHR, p-SAT5 and STAT5 in HepG2 cells transfected with GHR plasmid and/or siSTAT5 as indicated. (D) Relative mRNA level of RBP4 in the HepG2 cells transfected with GHR plasmid and/or siSTAT5 as indicated. (E) Western blots analysis of GHR, p-SAT5 and STAT5 in HepG2 cells transfected with siGHR and/or STAT5 plasmid as indicated. (F) Relative mRNA level of RBP4 in the HepG2 cells transfected with siGHR and/or STAT5 plasmid as indicated. (G) The HepG2 cells were cotransfected with different plasmids, then the luciferase activity was determined after 48 h-transfection (n=6). (H and I) ChIP assay was performed by using anti-STAT5 antibody. The elutes were analyzed by using primers for CCND1 STAT5 response elements (SRE), RBP4 SRE, or non-SRE region. The quantitative results were obtained by real-time PCR (H, n=6) or electrophoretic assay (I). Data are expressed as the mean ± SD. ns, no significant, *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test or one-way ANOVA).

Article Snippet: Moreover, the serum RBP4 concentrations were respectively carried out on humans (#DRB400) and mice (#DRBP40) using corresponding RBP4 Quantikine ELISA Kit (R&D Systems, USA).

Techniques: Western Blot, Infection, Binding Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction

Journal: Theranostics

Article Title: Growth hormone receptor disrupts glucose homeostasis via promoting and stabilizing retinol binding protein 4.

doi: 10.7150/thno.61192

Figure Lengend Snippet: Figure 7. GHR promotes RBP4 protein homeostasis through the HIF1α/TTR axis. (A) Western blots analysis of GHR, p-SAT5 and STAT5 in the livers of AAV-infected mice as indicated. (B) Relative mRNA levels of RBP4 in the livers of AAV-infected mice as indicated (n=6). (C) The concentrations of serum RBP4 of AAV-infected mice as indicated (n=6). (D) Elution profile of chylomicrons in the serum of AAV-GFP (left) or AAV-GHR (right) mice. Purified proteins were detected in column eluents by monitoring absorbance at 280 nm. (E) Western blots of RBP4 and TTR in serum of AAV-GFP or AAV-GHR mice, which were separated by gel filtration chromatography and collected according to the ultraviolet absorption peak of fractions. (F) Western blots analysis of HIF1α in the livers of AAV-infected mice as indicated. (G) The schematic representation of the HIF1α binding site in the promoter region of TTR. (H) Western blots analysis of GHR, TTR and HIF1α in the livers of AAV-infected mice as indicated. (I) The HepG2 cells were transfected with pGL-3 or TTR promoter reporter plasmid or 5x HIF1α response elements (HRE) reporter. After transfection for 24 h, the cells were exposed to a hypoxic condition for 24 h. Then the luciferase activity was determined (n=6). (J and K) ChIP assay was performed by using anti-HIF1α antibody. The elutes were analyzed by using primers for VEGF HRE, TTR HRE, or non-HRE region. The quantitative results were obtained by real-time PCR (J, n=6) or electrophoretic assay (K). Data are expressed as the mean ± SD. ns, no significant, *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test or one-way ANOVA).

Article Snippet: Moreover, the serum RBP4 concentrations were respectively carried out on humans (#DRB400) and mice (#DRBP40) using corresponding RBP4 Quantikine ELISA Kit (R&D Systems, USA).

Techniques: Western Blot, Infection, Purification, Filtration, Chromatography, Binding Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction