Journal: iScience
Article Title: ENT3: A lysosomal urate transporter regulating urate disposition and macrophage inflammation
doi: 10.1016/j.isci.2025.114249
Figure Lengend Snippet: Urate transport activity of ENT3 and ENT3-AA (A) Lysosomal urate accumulation was assessed by isolating lysosomes after cellular uptake of [ 14 C]urate (10 μM) for 30 min at pH 7.4 and 37°C in HEK293 cells transiently transfected with the plasmid encoding HA-SNBT1 (0.5 μg), with or without co-transfection of FLAG-ENT3 plasmid (0.5 μg). (B) Western blotting was performed using anti-FLAG and anti-HA antibodies on whole-cell lysates (10 μg protein per lane) prepared from HEK293 cells transiently expressing HA-SNBT1, with or without FLAG-ENT3. β-actin blots are shown as loading controls. (C) Immunofluorescence analysis revealed co-localization of HA-SNBT1 (green) with ATP1A1 (red), a plasma membrane marker, in transiently transfected HEK293 cells. Scale bars: 10 μm (D) Uptake of [ 14 C]urate (4 μM) was measured over 2 min at pH 5.0 and 37°C in HEK293 cells transiently expressing FLAG-ENT3 or FLAG-ENT3-AA, or mock cells. (E) Uptake of [ 3 H]adenosine (1 nM) was similarly assessed over 2 min at pH 5.0 and 37°C in the same cell groups. (F) Western blotting was performed using anti-FLAG antibody on whole-cell lysates (10 μg protein per lane) prepared from HEK293 cells transiently expressing FLAG-ENT3 or FLAG-ENT3-AA, or mock cells. β-actin blots are shown as loading controls. (G) Immunofluorescence images show the localization of FLAG-ENT3 and FLAG-ENT3-AA (green) in relation to ATP1A1 (red) in transiently transfected HEK293 cells. Scale bars: 10 μm. Data are presented as the mean ± SD from three biological replicates using separate cell preparations. Statistical significance was evaluated using two-way (A) or one-way (D and E) ANOVA followed by Bonferroni post-hoc test. ∗ p < 0.05 for the indicated comparison (A); ∗ p < 0.05 versus mock cells (D and E), # p < 0.05 versus FLAG-ENT3-expressing cells (D and E).
Article Snippet: Primary antibodies—mouse anti-FLAG monoclonal antibody, mouse anti-HA monoclonal antibody, and rabbit anti-ATP1A1 polyclonal antibody (Proteintech)—were applied at a dilution of 1:500 in PBS with 1% BSA and incubated for 1 h at room temperature.
Techniques: Activity Assay, Transfection, Plasmid Preparation, Cotransfection, Western Blot, Expressing, Immunofluorescence, Clinical Proteomics, Membrane, Marker, Comparison
Proteintech is a verified supplier 
![Urate transport activity of ENT3 and ENT3-AA (A) Lysosomal urate accumulation was assessed by isolating lysosomes after cellular uptake of [ 14 C]urate (10 μM) for 30 min at pH 7.4 and 37°C in HEK293 cells transiently transfected with the plasmid encoding HA-SNBT1 (0.5 μg), with or without co-transfection of FLAG-ENT3 plasmid (0.5 μg). (B) Western blotting was performed using <t>anti-FLAG</t> and anti-HA antibodies on whole-cell lysates (10 μg protein per lane) prepared from HEK293 cells transiently expressing HA-SNBT1, with or without FLAG-ENT3. β-actin blots are shown as loading controls. (C) Immunofluorescence analysis revealed co-localization of HA-SNBT1 (green) with ATP1A1 (red), a plasma membrane marker, in transiently transfected HEK293 cells. Scale bars: 10 μm (D) Uptake of [ 14 C]urate (4 μM) was measured over 2 min at pH 5.0 and 37°C in HEK293 cells transiently expressing FLAG-ENT3 or FLAG-ENT3-AA, or mock cells. (E) Uptake of [ 3 H]adenosine (1 nM) was similarly assessed over 2 min at pH 5.0 and 37°C in the same cell groups. (F) Western blotting was performed using anti-FLAG antibody on whole-cell lysates (10 μg protein per lane) prepared from HEK293 cells transiently expressing FLAG-ENT3 or FLAG-ENT3-AA, or mock cells. β-actin blots are shown as loading controls. (G) Immunofluorescence images show the localization of FLAG-ENT3 and FLAG-ENT3-AA (green) in relation to ATP1A1 (red) in transiently transfected HEK293 cells. Scale bars: 10 μm. Data are presented as the mean ± SD from three biological replicates using separate cell preparations. Statistical significance was evaluated using two-way (A) or one-way (D and E) ANOVA followed by Bonferroni post-hoc test. ∗ p < 0.05 for the indicated comparison (A); ∗ p < 0.05 versus mock cells (D and E), # p < 0.05 versus FLAG-ENT3-expressing cells (D and E).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_2747/pmc12752747/pmc12752747__gr1.jpg)
![Effects of genetic mutations on urate transport by ENT3-AA (A) Uptake of [ 14 C]urate (4 μM) by FLAG-tagged ENT3-AA (WT) and its mutants was evaluated over 2 min at pH 5.0 and 37°C in transiently transfected HEK293 cells. ND, not detected. (B) Western blotting was performed using anti-FLAG antibody on whole-cell lysates (10 μg protein per lane) prepared from transiently transfected HEK293 cells. β-actin blots are shown as loading controls. (C) Immunofluorescent imaging revealed the co-localization of FLAG-ENT3-AA (WT) and its mutants (green) with ATP1A1 (red), a plasma membrane marker, in transiently transfected HEK293 cells. Scale bars: 10 μm (D) Structural model of ENT3 was visualized using PyMOL based on AlphaFold predictions. The left and right panels show horizontal and extracellular side views, respectively. Amino acid residues altered by missense mutations, stop codon insertions, and frameshift mutations are shown in red, green, and blue, respectively. Data are presented as the mean ± SD from three biological replicates using separate cell preparations. (A) Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test. ∗ p < 0.05 versus control.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_2747/pmc12752747/pmc12752747__gr3.jpg)