Journal: The Journal of Clinical Investigation

Article Title: Endoplasmic reticulum–associated degradation is required for nephrin maturation and kidney glomerular filtration function

doi: 10.1172/JCI143988

Figure Lengend Snippet: (A–E) Representative confocal images of nephrin-KDEL (A), nephrin-ZO1 (B), podocin-KDEL (C), podocin-ZO1 (D), and CD2AP-ZO1 (E) in kidney sections from 3-week-old mice (n = 3 mice each). Arrows mark the perinuclear localization of nephrin (A), colocalization of nephrin with ZO1 (B), localization of podocin at the slit diaphragm (C), and colocalization of podocin and ZO1 (D) and CD2AP and ZO1 (E). Images with artificially enhanced KDEL signal are shown in Supplemental Figure 6A. (F) Western blot analysis of nephrin in kidney lysates from 3-, 5-, and 7-week-old mice and (G) quantitation of the percentage of b form nephrin in total nephrin. n = 10 mice each at 3 weeks; n = 10 Sel1Lfl/fl and n = 12 Sel1LPodCre at 5 weeks; and n = 4 each at 7 weeks. Seven-week-old Ire1aPodCre mice were included as a control (n = 3), and the original data are shown in Supplemental Figure 7A. Values represent the mean ± SEM. ***P < 0.001, by 2-tailed Student’s t test (3- and 5-week-old mice) and 1-way ANOVA (7-week-old mice). (H) Western blot analysis of nephrin in EndoH-treated kidney lysates from 5-week-old mice, with quantitation shown in Supplemental Figure 6D (n = 5 mice/group). r, EndoH-resistant form; s, EndoH-sensitive form.

Article Snippet: The following antibodies were used: SEL1L (Abcam, ab78298; 1:1000 for Western blotting); podocin (MilliporeSigma, P0372; 1:100 for immunostaining and ABclonal, A17337; 1:3000 for Western blotting); HRD1 (Proteintech, 13473-1-AP; 1:3000 for Western blotting); synaptopodin (Santa Cruz Biotechnology, sc-515842; 1:100 for immunostaining and ABclonal, A12049; 1:2000 for Western blotting); nephrin (ABclonal, A3048; 1:2000 for Western blotting, 1:100 for immunostaining); KDEL (Abcam, ab12223; 1:200 for immunostaining); ZO1 (Thermo Fisher Scientific, 33-9100; 1:100 for immunostaining); BiP (Abcam, ab21685; 1:5000 for Western blotting, 1:200 for immunostaining); CD2AP (Proteintech, 51046-1-AP; 1:300 for immunostaining); ubiquitin (Ub) (Santa Cruz Biotechnology, sc-8017; 1:1000 for Western blotting); Myc (MilliporeSigma, C3956; 1:5000 for Western blotting); Flag (MilliporeSigma, F-1804; 1:5000 for Western blotting); HSP90 (Santa Cruz Biotechnology, sc-13119; 1:5000 for Western blotting); and histone H2A (Cell Signaling Technology, 2578; 1:2000 for Western blotting).

Techniques: Western Blot, Quantitation Assay

Journal: The Journal of Clinical Investigation

Article Title: Endoplasmic reticulum–associated degradation is required for nephrin maturation and kidney glomerular filtration function

doi: 10.1172/JCI143988

Figure Lengend Snippet: (A) Western blot analysis following nephrin immunoprecipitation in kidney tissues from 5-week-old mice, showing the interaction between nephrin and BiP in the absence of ERAD. (B) Western blot analysis following HRD1 deletion in the HRD1–/– human podocyte line. CON, control. (C) Representative confocal images of nephrin and KDEL staining in human podocytes (n = 5 WT and n = 6 HRD1–/– cells). Scale bars: 5 μm. (D) Western blot analysis of nephrin in transfected WT and HRD1–/– HEK293T cells, digested with or without PNGase F (P) or EndoH (E), with quantitation of the percentage of EndoH-resistant and EndoH-sensitive forms shown below. (E) 35S pulse (30-min) chase (0, 1, 2, and 4 hours) analysis of nascent nephrin protein in HEK293T cells, and (F) quantitation of the percentage of a form nephrin in total nephrin. (G) Western blot analysis of Myc immunoprecipitates in transfected HEK293T cells, treated or not with 10 μM MG132 for 5 hours prior to harvesting, showing ERAD-mediated ubiquitination of nephrin. (H) Western blot analysis of nephrin protein decay in transfected HEK293T cells treated with brefeldin A and/or CHX for the indicated durations, with quantitation from 4 independent experiments shown below. (I) Western blot analysis of nephrin in transfected WT and Hrd1–/– N2a cells under nonreducing or reducing conditions, with the level of HMW nephrin normalized to total nephrin from 3 independent experiments shown below the blot. Data are representative of at least 3 independent experiments. Values represent the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-tailed Student’s t test.

Article Snippet: The following antibodies were used: SEL1L (Abcam, ab78298; 1:1000 for Western blotting); podocin (MilliporeSigma, P0372; 1:100 for immunostaining and ABclonal, A17337; 1:3000 for Western blotting); HRD1 (Proteintech, 13473-1-AP; 1:3000 for Western blotting); synaptopodin (Santa Cruz Biotechnology, sc-515842; 1:100 for immunostaining and ABclonal, A12049; 1:2000 for Western blotting); nephrin (ABclonal, A3048; 1:2000 for Western blotting, 1:100 for immunostaining); KDEL (Abcam, ab12223; 1:200 for immunostaining); ZO1 (Thermo Fisher Scientific, 33-9100; 1:100 for immunostaining); BiP (Abcam, ab21685; 1:5000 for Western blotting, 1:200 for immunostaining); CD2AP (Proteintech, 51046-1-AP; 1:300 for immunostaining); ubiquitin (Ub) (Santa Cruz Biotechnology, sc-8017; 1:1000 for Western blotting); Myc (MilliporeSigma, C3956; 1:5000 for Western blotting); Flag (MilliporeSigma, F-1804; 1:5000 for Western blotting); HSP90 (Santa Cruz Biotechnology, sc-13119; 1:5000 for Western blotting); and histone H2A (Cell Signaling Technology, 2578; 1:2000 for Western blotting).

Techniques: Western Blot, Immunoprecipitation, Staining, Transfection, Quantitation Assay