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. 2024 Oct 22;10:106. doi: 10.1038/s41421-024-00708-6

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a Illustration of the three uric acid transporter proteins containing YxNxxF motif. b-c Co-immunoprecipitation of NUMB and ABCG2. ABCG2-FLAG plasmid or empty vector was transient-transfected into HEK293 cells. Immunoprecipitation was done with FLAG antibody, followed by detecting the co-precipitation of ABCG2-FLAG with endogenous NUMB and ABCG2-FLAG and NUMB in input b. Star indicates non-specific band. On the other hand, NUMB-FLAG plasmid or empty vector was co-transfected with ABCG2-eGFP plasmid. Immunoprecipitation was done with FLAG antibody, followed by detecting the co-precipitation of ABCG2-eGFP with NUMB-FLAG and ABCG2-eGFP and NUMB-FLAG in input c. d Immunofluorescence staining of NUMB and ABCG2 in HEK293 cells. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Fluorescent signal intensity along the dashed lines are shown in the bottom panel. e Immunofluorescence staining of NUMB and ABCG2 in polarized MDCK cells. ABCG2-eGFP plasmid was transfected into MDCK cells. After reaching 100% confluence, the cells were maintained for another 3–5 days to establish a polarized monolayer. ZO-1 (blue), Na/K ATPase (magenta) and NUMB (red) were stained. NUMB was immuno-stained with rabbit anti-NUMB antibody (Cell signaling technology). Fluorescent signal intensity along lines in the amplified regions numbered with 1–3 are shown in the bottom panel. f Comparison of ABCG2-NUMB co-distribution among apical, lateral and basal side with One-way ANOVA with Tukey’s Multiple Comparison Test (n = 10 per group). g Immunofluorescence staining of NUMB, ABCG2 and early endosome marker EEA1, recycling endosome marker RAB11, late endosome marker RAB7 in HEK293 cells. Fluorescent signal intensity along the dashed lines are shown in h. i The statistic comparison of the triple positive dots per cells in HEK293 cells were carried out with One-way ANOVA with Tukey’s Multiple Comparison Test (n = 5 per group). j Co-distribution of ABCG2-eGFP (green), NUMB (blue) and RAB11 (red) in polarized MDCK cells. ZO-1 (cyan) was stained as an apical marker. k NUMB was immuno-stained with mouse anti-NUMB antibody (Santa Cruz). Fluorescent signal intensity along lines in the amplified regions numbered with 1–3 are shown. l The statistical comparison of the triple positive dots among in apical, lateral and basal domain were carried out with One-way ANOVA with Tukey’s Multiple Comparison Test (n = 10 per group). The results are presented as mean ± SD.