Journal: Free radical biology & medicine

Article Title: Adipocyte-secreted ANGPTL2 promotes hyperuricemia through inhibiting AKT/ABCG2 signaling.

doi: 10.1016/j.freeradbiomed.2025.03.048

Figure Lengend Snippet: Fig. 4. Angptl2 knockout improved renal UA excretion in HUA mice model (A) Angptl2 knockout and identification protocol (left panel), and genotype identification results by DNA agarose gel electrophoresis (right panel); (B) HUA mice model flow chart. (C) Fasting body weight before sacrifice. (D–G) Serum UA, CREA, BUN and XOD levels in WT, HUA, Angptl2 ko-HUA groups. (H) Urinary UA level in WT, HUA, Angptl2 ko-HUA groups. (I) Relative mRNA level of Abcg2, Slc22a8, Slc2a9 and Slc22a12 in the kidney. (J) Representative data of ABCG2 and GLUT9 in the kidney by Western blotting (left panel), and statistic data (right panel). ANGPTL2, angiopoietin-like protein 2; CREA, creatinine; BUN, blood urea nitrogen; UA, uric acid; HUA, hyperuricemia; ABCG2, ATP binding cassette subfamily G member 2; OAT3, organic anion transporter 3; GLUT9, glucose transporter 9; URAT1, urate transporter 1. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: After being deparaffinized, 0.5uM kidney sections were incubated overnight with the primary antibody ABCG2 (Abcam, USA) and GLUT9 (Proteintech, China) and stained the next day with the IHC kit (MXB Biotechnologies, China) and DAB (Proteintech, China) kit.

Techniques: Knock-Out, Agarose Gel Electrophoresis, Western Blot, Binding Assay

Journal: Free radical biology & medicine

Article Title: Adipocyte-secreted ANGPTL2 promotes hyperuricemia through inhibiting AKT/ABCG2 signaling.

doi: 10.1016/j.freeradbiomed.2025.03.048

Figure Lengend Snippet: Fig. 5. Adipocyte-specific overexpression of Angptl2 significantly increased serum UA levels in mice induced by adenine and potassium oxonate (A) HUA mice model flow chart. (B) Fasting body weight before sacrifice. (C) Serum ANGPTL2 levels after adipocyte-specific Angptl2 overexpression. (D–F) Serum CREA, BUN and UA levels after adipocyte-specific Angptl2 overexpression. (G) Relative mRNA level of Abcg2, Slc22a8, Slc2a9 and Slc22a12 in the kidney. (H) Representative data of ABCG2 and GLUT9 in the kidney by Western blotting (left panel), and statistic data (right panel). ANGPTL2, angiopoietin-like protein 2; CREA, creatinine; BUN, blood urea nitrogen; HUA, hyperuricemia; UA, uric acid; ABCG2, ATP binding cassette subfamily G member 2; OAT3, organic anion transporter 3; GLUT9, glucose transporter 9; URAT1, urate transporter 1. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: After being deparaffinized, 0.5uM kidney sections were incubated overnight with the primary antibody ABCG2 (Abcam, USA) and GLUT9 (Proteintech, China) and stained the next day with the IHC kit (MXB Biotechnologies, China) and DAB (Proteintech, China) kit.

Techniques: Over Expression, Western Blot, Binding Assay

Journal: Free radical biology & medicine

Article Title: Adipocyte-secreted ANGPTL2 promotes hyperuricemia through inhibiting AKT/ABCG2 signaling.

doi: 10.1016/j.freeradbiomed.2025.03.048

Figure Lengend Snippet: Fig. 6. ANGPTL2 decreased ABCG2 by suppressing AKT activity in HK2 cells (A) Flow chart of HK2 treatment groups. (B) UA levels in HK2 cell supernatants. (C) Relative mRNA level of Abcg2, Slc22a8, Slc2a9 and Slc22a12 in HK2 cells. (D) Representative data of ABCG2 and GLUT9 in HK2 cells by Western blotting (left panel), and statistic data (right panel). ANGPTL2, angiopoietin-like protein 2; rhA2, human recombinant proteins ANGPTL2; HUA, hyperuricemia; ABCG2, ATP binding cassette subfamily G member 2; OAT3, organic anion transporter 3; GLUT9, glucose transporter 9; URAT1, urate transporter 1. *p < 0.05, **p < 0.01, ****p < 0.0001.

Article Snippet: After being deparaffinized, 0.5uM kidney sections were incubated overnight with the primary antibody ABCG2 (Abcam, USA) and GLUT9 (Proteintech, China) and stained the next day with the IHC kit (MXB Biotechnologies, China) and DAB (Proteintech, China) kit.

Techniques: Activity Assay, Western Blot, Recombinant, Binding Assay

Journal: Free radical biology & medicine

Article Title: Adipocyte-secreted ANGPTL2 promotes hyperuricemia through inhibiting AKT/ABCG2 signaling.

doi: 10.1016/j.freeradbiomed.2025.03.048

Figure Lengend Snippet: Fig. 7. ANGPTL2 decreased ABCG2 by suppressing AKT activity in primary renal tubular epithelial cells. (A) Flow chart of RTECs treatment groups. (B) UA levels in RTECs supernatants. (C) Relative mRNA level of Abcg2, Slc22a8, Slc2a9 and Slc22a12 in RTECs. (D) Representative data of ABCG2 and GLUT9 in RTECs by Western blotting (left panel), and statistic data (right panel). ANGPTL2, angiopoietin-like protein 2; rhA2, human recombinant proteins ANGPTL2; HUA, hyperuricemia; ABCG2, ATP binding cassette subfamily G member 2; OAT3, organic anion transporter 3; GLUT9, glucose transporter 9; URAT1, urate transporter 1. *p < 0.05, **p < 0.01, ****p < 0.0001.

Article Snippet: After being deparaffinized, 0.5uM kidney sections were incubated overnight with the primary antibody ABCG2 (Abcam, USA) and GLUT9 (Proteintech, China) and stained the next day with the IHC kit (MXB Biotechnologies, China) and DAB (Proteintech, China) kit.

Techniques: Activity Assay, Western Blot, Recombinant, Binding Assay

Journal: Free radical biology & medicine

Article Title: Adipocyte-secreted ANGPTL2 promotes hyperuricemia through inhibiting AKT/ABCG2 signaling.

doi: 10.1016/j.freeradbiomed.2025.03.048

Figure Lengend Snippet: Fig. 9. Adipocyte-secreted ANGPTL2 promotes UA level through AKT/ABCG2 signaling in HK2 cells. (A) Flow diagram of C3H10 induction and treatment with FFA, as well as oil red O staining. (B) Representative data of ANGPTL2 after FFA treated C3H10 cells differentiated adipocytes by Western blotting (left panel), and statistic data (right panel). (C) ANGPTL2 levels in FFA-treated C3H10 cells differentiated adipocytes. (D) Flow chart of conditioned medium collected from C3H10 differ entiated into adipocytes treated with HK2 cells. (E) UA levels in HK2 cell supernatants after conditioned medium treatment. (F) Representative data of ABCG2 in HK2 cells by Western blotting (left panel), and statistic data (right panel). (G) Graphic summary of the study. Adipocyte-secreted ANGPTL2 decreased ABCG2 through inhibited AKT activation, then increased UA levels and led to HUA. ANGPTL2, angiopoietin-like protein 2; FFA, free fatty acid; HUA, hyperuricemia; ABCG2, ATP binding cassette G member 2; GLUT9, glucose transporter 9. *p < 0.05, **p < 0.01, ****p < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: After being deparaffinized, 0.5uM kidney sections were incubated overnight with the primary antibody ABCG2 (Abcam, USA) and GLUT9 (Proteintech, China) and stained the next day with the IHC kit (MXB Biotechnologies, China) and DAB (Proteintech, China) kit.

Techniques: Staining, Western Blot, Activation Assay, Binding Assay

Journal: Diabetes

Article Title: GLUT4 Is Not Necessary for Overload-Induced Glucose Uptake or Hypertrophic Growth in Mouse Skeletal Muscle

doi: 10.2337/db16-1075

Figure Lengend Snippet: Immunoblotting conditions

Article Snippet: Plasmids containing mouse GLUT1 (catalog #MR207871), GLUT3 (catalog #MR2097915), GLUT6 (catalog #MR219710), and GLUT10 (catalog #MR227535) and an HEK293 cell GLUT10 overexpression lysate (catalog #LY410718) were purchased from OriGene Technologies.

Techniques: Western Blot, Blocking Assay

Journal: PLoS ONE

Article Title: Expression, Purification, and Structural Insights for the Human Uric Acid Transporter, GLUT9, Using the Xenopus laevis Oocytes System

doi: 10.1371/journal.pone.0108852

Figure Lengend Snippet: (A) Expression level of hGLUT9b by Western blot analysis. (B) Surface expression of hGLUT9b as determined by deglycosylation analysis using PNGase of fully denatured DDM based total lysate. (C) Surface biotinylation and pull-down reveals a highly enriched hGLUT9b surface membrane fraction.

Article Snippet: cDNA from hGLUT9b ( Gene ID: 56606 ) was purchased from Origene and sub-cloned by PCR into the pMJB08 expression vector using forward primers 5′ CAT TTC TCG AGA TGA AGC TCA GTA AAA AGG ACC GAG G′ and reverse primers 5′ TTA ATT CTA GAT TAA GGC CTT CCA TTT ATC TTA CCA TC 3′ .

Techniques: Expressing, Western Blot, Membrane

Journal: PLoS ONE

Article Title: Expression, Purification, and Structural Insights for the Human Uric Acid Transporter, GLUT9, Using the Xenopus laevis Oocytes System

doi: 10.1371/journal.pone.0108852

Figure Lengend Snippet: (A) Water injected controls and hGLUT9b expressing oocytes were clamped at −30 mV and exposed for 30 sec to 500 µM urate containing OR2 medium. Current evoked by urate averaged 70+/−10 nA (n>20). (B) Same experiment as outlined above, but with application of the non-specific GLUT9 inhibitor, phloretin. Oocyte were clamped at −30 mV and exposed for 15 sec to 500 µM urate followed by 20 sec of 500 µM urate +25 µM Phloretin. Phloretin inhibited uric acid current by 45±4% (n = 3).

Article Snippet: cDNA from hGLUT9b ( Gene ID: 56606 ) was purchased from Origene and sub-cloned by PCR into the pMJB08 expression vector using forward primers 5′ CAT TTC TCG AGA TGA AGC TCA GTA AAA AGG ACC GAG G′ and reverse primers 5′ TTA ATT CTA GAT TAA GGC CTT CCA TTT ATC TTA CCA TC 3′ .

Techniques: Injection, Expressing

Journal: PLoS ONE

Article Title: Expression, Purification, and Structural Insights for the Human Uric Acid Transporter, GLUT9, Using the Xenopus laevis Oocytes System

doi: 10.1371/journal.pone.0108852

Figure Lengend Snippet: (A) Silver-stained and (B) Western blot SDS/polyacrylamide gels show that recombinant human GLUT9b runs at ∼60 kDa, corresponding to the expected molecular weight. Oligomers are observed at ∼120 and 200 kDa. First line corresponds to the pellet fraction at 5,000 g after membrane solubilization (P5000) and the supernatant was loaded on the IMAC column (Input). The Western blot using anti-HA shows that all hGLUT9b was extracted and solubilized from the membrane fraction. The three following lanes correspond to the unbound and washed fractions and demonstrate that hGLUT9b binds to the column. The protein was eluted using HRV 3C protease at the PreScission site.

Article Snippet: cDNA from hGLUT9b ( Gene ID: 56606 ) was purchased from Origene and sub-cloned by PCR into the pMJB08 expression vector using forward primers 5′ CAT TTC TCG AGA TGA AGC TCA GTA AAA AGG ACC GAG G′ and reverse primers 5′ TTA ATT CTA GAT TAA GGC CTT CCA TTT ATC TTA CCA TC 3′ .

Techniques: Staining, Western Blot, Recombinant, Molecular Weight, Membrane

Journal: PLoS ONE

Article Title: Expression, Purification, and Structural Insights for the Human Uric Acid Transporter, GLUT9, Using the Xenopus laevis Oocytes System

doi: 10.1371/journal.pone.0108852

Figure Lengend Snippet: (A) Superose 6 gel filtration chromatography shows two peaks. The first peak corresponds to the dead volume of the gel filtration where large molecular weight proteins or aggregates do not interact with the beads and quickly pass through the column. The second peak corresponds principally to the monomeric (sharp peak, right side) and oligomeric (shoulder, left side) states. Western blot representation of fractions 30 to 43 after gel filtration showing clearly the separation of the second peak into hGLUT9b oligomers and monomer. (B) Silver-staining of hGLUT9b monomers obtained from fraction 35. (C) Micrograph of hGLUT9 particles obtained from negatively-stained TEM. Scale bar is 50 nm. Gallery representation of the 9 class-averages obtained from 1439 particles. Each representation is scaled to a 26 nm square.

Article Snippet: cDNA from hGLUT9b ( Gene ID: 56606 ) was purchased from Origene and sub-cloned by PCR into the pMJB08 expression vector using forward primers 5′ CAT TTC TCG AGA TGA AGC TCA GTA AAA AGG ACC GAG G′ and reverse primers 5′ TTA ATT CTA GAT TAA GGC CTT CCA TTT ATC TTA CCA TC 3′ .

Techniques: Filtration, Chromatography, Molecular Weight, Western Blot, Silver Staining, Staining

Journal: Fluids and Barriers of the CNS

Article Title: Immunohistochemical and in situ hybridization study of urate transporters GLUT9/URATv1, ABCG2, and URAT1 in the murine brain

doi: 10.1186/s12987-016-0046-x

Figure Lengend Snippet: Immunofluorescence staining of GLUT9 in PFA-fixed murine brain sections. Frozen sections of paraformaldehyde-fixed wild-type murine brain were used for immunofluorescence staining. a , b Antigen absorption test. Immunofluorescence staining of the ependymal wall of the dorsal third ventricle using a anti-GLUT9 antibody and b antigen-preabsorbed antibody. Scale bar 100 µm. c , d Immunofluorescence staining of GLUT9 ( magenta ), acetylated-tubulin (Ac-Tubulin, green ) and DAPI ( blue ) on ependymal cells. Scale bar 10 µm. e – g Immunofluorescence staining of GLUT9 ( magenta ) and NeuN ( green ) showing co-localization in neurons. Scale bar 10 µm. D3V, dorsal third ventricle; DAPI, 4′,6-diamidino-2-phenylindole; NeuN, neuronal nucleus marker

Article Snippet: For antigen absorption experiment, GLUT9 peptide (NBP1-05054PEP, Novus Biologicals) was used at a concentration of 100 μg/ml.

Techniques: Immunofluorescence, Staining, Marker

Journal: Fluids and Barriers of the CNS

Article Title: Immunohistochemical and in situ hybridization study of urate transporters GLUT9/URATv1, ABCG2, and URAT1 in the murine brain

doi: 10.1186/s12987-016-0046-x

Figure Lengend Snippet: GLUT9/URATv1 immunoreactivity is detected in ependymal cells of all ventricles. Frozen sections of paraformaldehyde-fixed wild-type murine brain were used for immunofluorescence staining. The red squares in the diagrams indicate the region shown in each image. a – f GLUT9 staining of coronal sections. b – d Images were obtained from the same section. Scale bar 100 µm. LV, lateral ventricle; V3V, ventral third ventricle; AQ, aqueduct; 4V, fourth ventricle

Article Snippet: For antigen absorption experiment, GLUT9 peptide (NBP1-05054PEP, Novus Biologicals) was used at a concentration of 100 μg/ml.

Techniques: Immunofluorescence, Staining

Journal: Fluids and Barriers of the CNS

Article Title: Immunohistochemical and in situ hybridization study of urate transporters GLUT9/URATv1, ABCG2, and URAT1 in the murine brain

doi: 10.1186/s12987-016-0046-x

Figure Lengend Snippet: Immunohistochemistry and immunofluorescence staining of GLUT9 in methacarn-fixed murine brain. Paraffin sections of methacarn-fixed murine brain were used for immunostaining. a , b Antigen absorption test. Immunohistochemistry using showing a anti-GLUT9 antibody staining in the third ventricle ependyma and parenchyma and b antigen-preabsorbed antibody. Scale bar 100 µm. c GLUT9 immunoreactivity observed in brain capillaries in the cortex. Scale bar 100 µm. d – f Immunofluorescence staining of d GLUT9 and e P-gp showing co-localization in capillaries. Blue indicates DAPI-stained nucleus of the brain capillary endothelial cell. Scale bar 10 µm. P-gp, P-glycoprotein

Article Snippet: For antigen absorption experiment, GLUT9 peptide (NBP1-05054PEP, Novus Biologicals) was used at a concentration of 100 μg/ml.

Techniques: Immunohistochemistry, Immunofluorescence, Staining, Immunostaining

Journal: Fluids and Barriers of the CNS

Article Title: Immunohistochemical and in situ hybridization study of urate transporters GLUT9/URATv1, ABCG2, and URAT1 in the murine brain

doi: 10.1186/s12987-016-0046-x

Figure Lengend Snippet: Immunofluorescence staining of ABCG2 in methanol/acetone-fixed and methacarn-fixed murine brain. Fresh frozen sections of the dorsal third ventricle were prepared from wild-type (WT) and ABCG2 knockout (KO) mice and post-fixed with methanol and acetone. Immunofluorescence staining of ABCG2 was seen in choroid plexus and capillaries in sections from a WT but not in b ABCG2 KO mouse. Scale bar 100 µm. c Immunofluorescence staining of ABCG2 ( magenta ) and GLUT9 ( green ) showing ABCG2 in the choroid plexus and GLUT9 in the ependyma. Scale bar 100 µm. d – f Immunofluorescence staining of d ABCG2 and e GLUT9 using methacarn-fixed paraffin section of capillaries showing co-localization in f . Blue indicates DAPI-stained nucleus of the brain capillary endothelial cell. Scale bar 10 µm. CP, choroid plexus; D3V, dorsal third ventricle

Article Snippet: For antigen absorption experiment, GLUT9 peptide (NBP1-05054PEP, Novus Biologicals) was used at a concentration of 100 μg/ml.

Techniques: Immunofluorescence, Staining, Knock-Out, Paraffin Section

Journal: Fluids and Barriers of the CNS

Article Title: Immunohistochemical and in situ hybridization study of urate transporters GLUT9/URATv1, ABCG2, and URAT1 in the murine brain

doi: 10.1186/s12987-016-0046-x

Figure Lengend Snippet: Localization of mRNA of urate transporters in murine brain by fluorescence in situ hybridization. a – c Merged bright field and fluorescence images are seen in the upper panels , while the lower panels show the fluorescence images alone. Green dots show Fast Blue signal observed with the Alexa 750 filter set, while the red dots show Fast Red signal observed with the Cy3 filter set. mRNA probes: Slc22a12 (URAT1), Slc2a9 (GLUT9), Abcg2 (ABCG2). Slc22a12 is expressed largely in the ependyma and less in the parenchyma, Slc2a9 is expressed in the ependyma and parenchyma and Abcg2 in the choroid plexus epithelium

Article Snippet: For antigen absorption experiment, GLUT9 peptide (NBP1-05054PEP, Novus Biologicals) was used at a concentration of 100 μg/ml.

Techniques: Fluorescence, In Situ Hybridization