Journal: Poultry Science

Article Title: LncRNA HAND2OT encoding micropeptide regulates the estradiol secretion, proliferation and apoptosis of ovarian follicular theca cells in chickens

doi: 10.1016/j.psj.2025.106206

Figure Lengend Snippet: The lncRNA HAND2OT encodes a micropeptide. (A) The predicted results of protein-coding ability of HAND2OT by CNCI and CPC2. (B) Schematic diagram of the ORF of HAND2OT predicted based on NCBI ORF Finder. Construction diagrams of the fusion vectors used to verify protein encoding (C, F, I). In pEGFP-MUT and ORF1/2/3+pEGFP-MUT vectors, the initiation codon (ATG) of EGFP and the termination codon (TAA/TGA) of HAND2OT ORF1/2/3 were deleted (C), and the ORF2-MUT vector was constructed based on the ORF2+pEGFP-MUT vector by mutating the start codon (ATG) of ORF2 to ATT (F). In ORF1/2/3-3 × Flag vectors, the termination codon (TAA/TGA) of HAND2OT ORF1/2/3 were deleted (I). Fluorescence imaging and western blotting demonstrating EGFP expression in chicken theca cells (D, E, G, H). The scale bars are 200 μm. (J) Western blotting demonstrating Flag protein expression in chicken theca cells. (K) Schematic illustration of the predicted amino acid sequence of the HAND2OT-66aa micropeptide encoded by ORF2.

Article Snippet: The PVDF membrane was incubated with anti-EGFP tag mouse mAb (Engibody, Shanghai, China) at 1:6000 dilution, anti-FLAG tag mouse mAb (Engibody, Shanghai, China) at 1:4000 dilution, and mouse anti-GAPDH monoclonal antibody (Proteintech, Wuhan, China) at 1:50,000 dilution.

Techniques: Plasmid Preparation, Construct, Fluorescence, Imaging, Western Blot, Expressing, Sequencing

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

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: 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.

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: Transfection, Western Blot, Imaging, Clinical Proteomics, Membrane, Marker, Control