Journal: Cellular and Molecular Life Sciences

Article Title: S100A16 promotes acute kidney injury by activating HRD1-induced ubiquitination and degradation of GSK3β and CK1α

doi: 10.1007/s00018-022-04213-5

Figure Lengend Snippet: HRD1 involves in the activation of Wnt/β-catenin signaling pathway and is regulated by S100A16 in injured renal fibroblasts. a Representative micrographs in the corticomedullary junction of mice kidneys showed the expression of HRD1 in WT mice and S100A16 +/− mice at 1 day after IRI, as determined by immunohistochemical staining. Scale bar, 50 μm, 20 μm (Enlarged). b HRD1, GSK3β and CK1α expressions were tested using kidney tissues from WT mice and S100A16 +/− mice at 1 day after IRI compared with sham mice by western blot assays. c Quantitation of immunoblot data for HRD1, GSK3β and CK1α proteins as in b . **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05. n = 3; each point represents the expression in a sample pooled from two mice. d Western blot analyses showed that ICG-001 blocked the increased HRD1 expression induced by H/R in NRK-49F cells, and ICG-001 also recovered the expressions of GSK3β and CK1α. Cell lysates after various treatments, as indicated, were immunoblotted with antibodies against HRD1, GSK3β, CK1α and β-actin. e Quantitation of western blot data for HRD1, GSK3β and CK1α proteins as in d . *** P < 0.001, ** P < 0.01, versus control; ## P < 0.01, # P < 0.05, n.s. not significant, versus H/R alone. n = 3. f Western blot analyses showed that knockdown of S100A16 inhibited the HRD1 expression and enhanced the expressions of GSK3β and CK1α in normal or hypoxia condition. g Quantitation of western blot data for HRD1, GSK3β and CK1α proteins as in f . *** P < 0.001, ** P < 0.01, versus scrambled shRNA; ## P < 0.01, versus H/R + scrambled shRNA. n = 3. h Western blots showed that overexpression of S100A16 increased the HRD1 expression, but it was impeded by ICG-001 in NRK-49F cells. The GSK3β and CK1α expressions were opposite to HRD1. i Quantified HRD1, GSK3β and CK1α protein levels in h . ** P < 0.01, versus pcDNA3.1; # P < 0.05, versus S100A16 OE. n = 3

Article Snippet: The primary antibodies against S100A16 (HPA045841; Sigma-Aldrich, St Louis, MO, USA), β-catenin (610153; BD Biosciences, San Jose, CA, USA), HRD1 (13473-1-AP; Proteintech, Chicago, IL, USA) were used with 1:100 dilution.

Techniques: Activation Assay, Expressing, Immunohistochemical staining, Staining, Western Blot, Quantitation Assay, Control, Knockdown, shRNA, Over Expression

Journal: Cellular and Molecular Life Sciences

Article Title: S100A16 promotes acute kidney injury by activating HRD1-induced ubiquitination and degradation of GSK3β and CK1α

doi: 10.1007/s00018-022-04213-5

Figure Lengend Snippet: HRD1 degrades both GSK3β and CK1α via the ubiquitin–proteasome pathway in NRK-49F cells. a The interaction between HRD1 and either GSK3β or CK1α was detected in the co-IP analysis in NRK-49F cells. b A cycloheximide chase was performed to establish the time course of GSK3β or CK1α biogenesis. NRK-49F cells were infected with or without Ad-HRD1 for 48 h, and then the cells were treated with CHX (100 μg/ml) for 0, 2,4 or 6 h. The expressions of GSK3β and CK1α in whole-cell lysates were measured by western blotting. c Quantitation of western blot data for GSK3β and CK1α proteins as in b . * P < 0.05. n = 3. d The presence of MG132 (20 µM) increased the expressions of GSK3β and CK1α with or without Ad-HRD1 infection compared with controls in NRK-49F cells. e Quantification of GSK3β and CK1α protein expressions from experiments as shown in d . *** P < 0.001, ** P < 0.01, * P < 0.05. n = 3. f More ubiquitin conjugated to GSK3β was detected in the cells overexpressing HRD1 compared with no HRD1 transfection. g More ubiquitin conjugated to CK1α was detected in the cells overexpressing HRD1 compared with no HRD1 transfection. h Quantification of ubiquitin conjugated to GSK3β as in f , normalized to GAPDH expression. *** P < 0.001. n = 3. i Quantification of ubiquitin conjugated to CK1α as in g , normalized to GAPDH expression. ** P < 0.01. n = 3

Article Snippet: The primary antibodies against S100A16 (HPA045841; Sigma-Aldrich, St Louis, MO, USA), β-catenin (610153; BD Biosciences, San Jose, CA, USA), HRD1 (13473-1-AP; Proteintech, Chicago, IL, USA) were used with 1:100 dilution.

Techniques: Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Infection, Western Blot, Quantitation Assay, Transfection, Expressing

Journal: Cellular and Molecular Life Sciences

Article Title: S100A16 promotes acute kidney injury by activating HRD1-induced ubiquitination and degradation of GSK3β and CK1α

doi: 10.1007/s00018-022-04213-5

Figure Lengend Snippet: S100A16 down-regulates the expressions of both GSK3β and CK1α by HRD1 to affect downstream HGF in NRK-49F cells. a Western blot analyses showed that overexpression of S100A16 aggravated the downregulated expressions of GSK3β, CK1α, and HGF induced by Ad-HRD1 in NRK-49F cells. b Quantitation of western blot data for GSK3β, CK1α, and HGF proteins, as in a . * P < 0.05, versus pcDNA3.1; ## P < 0.01, versus pcDNA3.1 + Ad-HRD1. n = 3. c Real-time qPCR demonstrated that overexpression of S100A16 aggravated the reduction of relative HGF/β-actin mRNA level induced by Ad-HRD1 compared with the Ad-HRD1 treatment alone in NRK-49F cells. ** P < 0.01, versus pcDNA3.1; # P < 0.05, versus pcDNA3.1 + Ad-HRD1. n = 3. d Western blot analyses showed that knockdown S100A16 recovered Ad-HRD1-induced the down-regulated expressions of GSK3β, CK1α, and HGF. e Quantitation of western blot data for GSK3β, CK1α and HGF proteins, as in d . ** P < 0.01, * P < 0.05, versus scrambled shRNA; ## P < 0.01, versus scrambled shRNA + Ad-HRD1. n = 3. f Representative images showed the accumulation of β-catenin fluorescence in the nucleus of HRD1-overexpressing NRK-49F cells. The β-catenin fluorescence was amplified after overexpressing S100A16 in the HRD1-overexpressing NRK-49F cells. Scale bar, 20 μm

Article Snippet: The primary antibodies against S100A16 (HPA045841; Sigma-Aldrich, St Louis, MO, USA), β-catenin (610153; BD Biosciences, San Jose, CA, USA), HRD1 (13473-1-AP; Proteintech, Chicago, IL, USA) were used with 1:100 dilution.

Techniques: Western Blot, Over Expression, Quantitation Assay, Knockdown, shRNA, Fluorescence, Amplification

Journal: Cellular and Molecular Life Sciences

Article Title: S100A16 promotes acute kidney injury by activating HRD1-induced ubiquitination and degradation of GSK3β and CK1α

doi: 10.1007/s00018-022-04213-5

Figure Lengend Snippet: The Wnt/β-catenin signaling activation induced by S100A16–HRD1–GSK3β/CK1α pathway in renal fibroblasts under IRI condition promotes the occurrence of AKI. Schematic diagram shows the increased expression of S100A16 in fibroblasts during renal ischemia and hypoxia. The expression of the E3 ubiquitin ligase HRD1 is elevated, and the members of β-catenin degradation complex, GSK3β and CK1α, are destroyed via the ubiquitin–proteasome pathway, thereby promoting the accumulation of β-catenin and its transfer to the cell nucleus. Activation of the Wnt/β-catenin signaling pathway then inhibits the transcription of the downstream HGF that secreted by fibroblasts, and this procedure eventually leads to unrepairable kidney injury and renal dysfunction

Article Snippet: The primary antibodies against S100A16 (HPA045841; Sigma-Aldrich, St Louis, MO, USA), β-catenin (610153; BD Biosciences, San Jose, CA, USA), HRD1 (13473-1-AP; Proteintech, Chicago, IL, USA) were used with 1:100 dilution.

Techniques: Activation Assay, Expressing, Ubiquitin Proteomics

Journal: Scientific Reports

Article Title: PGC-1α attenuates hydrogen peroxide-induced apoptotic cell death by upregulating Nrf-2 via GSK3β inactivation mediated by activated p38 in HK-2 Cells

doi: 10.1038/s41598-017-04593-w

Figure Lengend Snippet: The involvement of p38/GSK3β/Nrf-2 axis for cytoprotective effects of PGC-1α. To understand the molecular mechanism for cytoprotective effects of PGC-1α, the activation of Keap1-, GSK3β- or Hrd1 ( A ) and MAPKs ( B ), as an upstream signal molecules of Nrf-2, were compared to Mock and PGC-1α cells for indicated time points after H 2 O 2 treatment. To further elucidate the specificity of p38-mediated signal cascade for cytoprotective effects of PGC-1α cells, p38 specific effects were analyzed by western blotting ( C ) and MTT assay ( D ) in PGC-1α cells treated with H 2 O 2 in the presence or absence of p38 inhibitor (SB203580) for 2 h or 4 h, respectively. Inhibitor of ERK1/2 (PD98059) was used as non-effective control on PGC-1α effect. Activation of p38 or inactivation of GSK3β was checked with phosphor specific antibody at Thr180/Tyr182 residue or at Ser9 residue, respectively. Full-length blots of each tested protein are reported in Supplementary Figure . Error bars denote the mean ± S.D. of triplicate samples. MTT assay was performed to n = 7. * p < 0.05 H 2 O 2 -untreated Mock vs. PGC-1α at indicated time points; # p < 0.05; p38 inhibitor treated vs. untreated; † p < 0.05; ERK1/2 inhibitor treated vs. untreated.

Article Snippet: Antibody against Hrd1 was purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Activation Assay, Western Blot, MTT Assay, Control, Residue

Journal: PLoS Pathogens

Article Title: Cytolethal Distending Toxins Require Components of the ER-Associated Degradation Pathway for Host Cell Entry

doi: 10.1371/journal.ppat.1004295

Figure Lengend Snippet: (a) Co-immunoprecipitation of Derl2 and Hrd1. Derl2 was immunoprecipitated as in and samples were analyzed for Hrd1 by western blot. (b) CRISPR mediated deletion of Hrd1 (ΔHrd1) results in decreased expression as judged by western blot of Hrd1 from α-Hrd1 immunoprecipitated protein from normalized cell lysates. (c) Co-immunoprecipitation of Derl2 with Hrd1. Hrd1 was immunoprecipitated and samples were analyzed for Derl2 by western blot. (d–g) Wild type 293 and ΔHrd1 cells were intoxicated with Aa-CDT (d), Hd-CDT (e), Ec-CDT (f) and Cj-CDT (g) similar to . Percent viability is normalized to unintoxicated controls and error bars indicate standard error. (h–j) Retrograde trafficking of Hd-CDT in ΔHrd1 cells is blocked at the endoplasmic reticulum. pDsRed2-ER (red) transfected 293 cells and ΔHrd1 cells were incubated with Hd-CDT on ice, washed and incubated at 37°C for 240 minutes. Cells were then fixed and stained with DAPI (nuclei, blue) and α-Hd-CdtB (green) antibody. White scale bars indicate 5 µm. (i,j) Quantification of microscopy results comparing the percentage of cells with at least one green puncta localized to the nucleus (i), or Pearson's coefficient values indicating colocalization of the Hd-CdtB signal with the ER (j). Images and quantitation are representative of those collected from a total of 30 randomly chosen cells analyzed during two independent experiments and error bars represent standard deviations. Unless otherwise noted, data are representative of at least three independent experiments.

Article Snippet: Membranes were probed with either rabbit anti-Derl2 antibody (Sigma Aldrich) or rabbit anti-Hrd1 polyclonal antibody (Novus Biologicals) at a 1∶2000 dilution followed by HRP conjugated α-rabbit antibody (Invitrogen) to allow detection.

Techniques: Immunoprecipitation, Western Blot, CRISPR, Expressing, Transfection, Incubation, Staining, Microscopy, Quantitation Assay

Journal: PLoS Pathogens

Article Title: Cytolethal Distending Toxins Require Components of the ER-Associated Degradation Pathway for Host Cell Entry

doi: 10.1371/journal.ppat.1004295

Figure Lengend Snippet: (a) Derl2-GFP fails to bind p97, similar to Derl2ΔC. 293 cells were transfected with vectors encoding S-tagged versions of the indicated forms of Derl2. After 3 days, the cells were lysed and western blot was performed on S-protein precipitates with anti-p97 and anti-S-tag antibodies (b) Overexpression of Derl2-GFP does not affect Hd-CDT intoxication of parental A745TKR cells. Parental A745TKR cells expressing empty vector, Derl2 or Derl2-GFP were intoxicated with Hd-CDT, similar to . (c, d) Derl2-GFP and Derl2ΔC complement sensitivity to Hd-CDT in CHO-CDT R C1. CHO-CDT R C1 cells expressing empty vector, Derl2, (c) Derl2-GFP or (d) Derl2ΔC were intoxicated similar to . (e) Dominant negative p97 reduces sensitivity of 293 cells to Hd-CDT. 293 cells stably expressing TCRαGFP were transfected with plasmids encoding CD4 and either dominant negative (R586A) or control (R700A) p97, followed by intoxication with Hd-CDT for 48 hours and staining with Hoechst and anti-CD4 antibodies. Flow cytometry was performed to obtain geometric mean fluorescence values for TCRαGFP (GFP) in CD4+ cells and cell cycle profile of CD4 negative (grey shaded; untransfected control) and CD4 positive cells (black lines). (f) The Derl2 WR motif is not required for intoxication by Hd-CDT. CHO-CDT R C1 cells expressing empty vector, wildtype Derl2, Derl2 Q53A, Derl2 W55A or Derl2 T59A were intoxicated similar to . (g–i) Retrograde trafficking of Hd-CDT in p97 deficient cells is blocked at the endoplasmic reticulum. (g) Following transfection with pH2B-GFP (blue) and either dominant negative or control p97, wildtype and ΔHrd1 cells were incubated with Hd-CDT on ice, washed and incubated at 37°C for 240 minutes. Cells were then fixed and stained with anti-Hd-CdtB (green) antibody and anti-calreticulin antibody (red). White scale bars indicate 5 µm. pH2B-GFP pseudo-colored blue; Hd-CdtB pseudo-colored green and calreticulin pseudo-colored red (h, i) Quantification of microscopy results comparing the percentage of cells with at least one green puncta localized to the nucleus or Pearson's coefficient values indicating colocalization of the Hd-CdtB signal with the ER. Images and quantitation are representative of those collected from a total of 30 randomly chosen cells analyzed during two independent experiments and error bars represent standard deviations. Unless otherwise noted, data are representative of at least three independent experiments, percent viability is normalized to unintoxicated controls and error bars indicate standard error.

Article Snippet: Membranes were probed with either rabbit anti-Derl2 antibody (Sigma Aldrich) or rabbit anti-Hrd1 polyclonal antibody (Novus Biologicals) at a 1∶2000 dilution followed by HRP conjugated α-rabbit antibody (Invitrogen) to allow detection.

Techniques: Transfection, Western Blot, Over Expression, Expressing, Plasmid Preparation, Dominant Negative Mutation, Stable Transfection, Control, Staining, Flow Cytometry, Fluorescence, Incubation, Microscopy, Quantitation Assay

Journal: PLoS Pathogens

Article Title: Cytolethal Distending Toxins Require Components of the ER-Associated Degradation Pathway for Host Cell Entry

doi: 10.1371/journal.ppat.1004295

Figure Lengend Snippet: (a) Derl2 deficiency causes resistance to ricin. A745TKR cells, CHO-CDT R C1 cells, and CHO-CDT R C1 cells expressing Derl2 were seeded in a 384-well plate (1×10 3 cells/well) and allowed to adhere overnight, followed by 48 hour intoxication with ricin and quantitation of viability using ATPlite 1-step reagent (Perkin Elmer). Ricin LD 50 values were calculated from three independent experiments and paired t-test was performed to calculate two tailed p-values. (b) CRISPR mediated Hrd1 deletion in 293 cells causes resistance to ricin. Wildtype and Hrd1-deleted 293 cells were intoxicated with ricin, similar to figure (a). (c) Derl2ΔC complements the resistance to ricin. CHO-CDT R C1 cells expressing empty vector, Derl2 and Derl2ΔC were intoxicated similar to (a). (d) The Derl2 WR motif is not required for intoxication by ricin. CHO-CDT R C1 cells expressing empty vector, wildtype Derl2, Derl2 Q53A, Derl2 W55A and Derl2 T59A were intoxicated similar to (a). Data are representative of at least three independent experiments performed in triplicate, percent viability is normalized to unintoxicated controls and error bars indicate standard error.

Article Snippet: Membranes were probed with either rabbit anti-Derl2 antibody (Sigma Aldrich) or rabbit anti-Hrd1 polyclonal antibody (Novus Biologicals) at a 1∶2000 dilution followed by HRP conjugated α-rabbit antibody (Invitrogen) to allow detection.

Techniques: Expressing, Quantitation Assay, Two Tailed Test, CRISPR, Plasmid Preparation

Journal: Frontiers in Veterinary Science

Article Title: The H protein of attenuated canine distemper virus is degraded via endoplasmic reticulum-associated protein degradation

doi: 10.3389/fvets.2023.1214318

Figure Lengend Snippet: The degradation of CDV 851H protein via ERAD. (A) The inhibition of ERAD by Eevarestatin I increased the CDV 851H protein level. 293T cells were transfected with Flag-CDV H for 24 h, then were treated with Eevarestatin I (4 μM) for 4 h. The cell lysates were analyzed by Western blot using anti-Flag and anti-GAPDH antibodies. The gray values of protein bands were analyzed by ImageJ software and the ratio of target protein gray values to GAPDH was calculated. (B) Knockdown of Hrd1 increased the CDV 851H protein level. After transfecting 293T cells with siRNA for Hrd1 or si-NC for 24 h, the cells were transfected with H protein of CDV 851 and CDV NJ(11)2 for another 24 h. Western blot was used to detect the CDV H protein level and confirm the silencing efficacy of Hrd1-targeted siRNA. The gray values of protein bands were analyzed by ImageJ software and the ratio of target protein gray values to GAPDH was calculated. All results are presented as means ± SD obtained from at least three independent sample preparations. * p < 0.05, ** p < 0.01.

Article Snippet: The antibodies were obtained commercially: anti-Flag mouse monoclonal antibody (F1804, Sigma), anti-ATF6 rabbit polyclonal antibody (DF6009, Affinity), anti-Hrd1 rabbit polyclonal antibody (A2605, Abclonal), anti-HA mouse monoclonal antibody (BD-PM2095, Biodragon), anti-GAPDH mouse monoclonal antibody (60004-1-Ig, Proteintech), HRP-conjugated goat anti-mouse IgG (BF03001, Biodragon), and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (A0568, Beyotime).

Techniques: Inhibition, Transfection, Western Blot, Software, Knockdown

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: Representative confocal images of SEL1L (A) and HRD1 (B) costaining with WT1 in kidney tissues from healthy humans. Asterisks identify WT1+ podocytes. Arrowheads indicate distal tubular cells also expressing SEL1L and HRD1. In addition to expression in podocytes, WT1 was also expressed in parietal epithelial cells lining the Bowman capsule (arrows). Scale bars: 100 μm, 20 μm, and 10 μm.

Article Snippet: The following antibodies were used for immunofluorescence of human samples: SEL1L ( 43 ) (home-made {"type":"entrez-nucleotide","attrs":{"text":"E12049","term_id":"22027566","term_text":"E12049"}} E12049 , specific for both human and mouse, rabbit, 1:500); HRD1 (provided by Richard Wojcikiewicz, SUNY Upstate Medical University, Syracuse, New York, USA; rabbit, 1:50 for immunostaining); and WT1–Alexa Fluor 647 (Abcam, ab202639, 1:500). scRNA-Seq of the human kidney.

Techniques: Expressing

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–C) Representative confocal images of SEL1L (A) and HRD1 (B) costaining with WT1 in kidney tissues from 3-week-old Sel1Lfl/fl and Sel1LPodCre mice (n = 3 mice each), with quantitation shown in C (n = 59, 45, 130, and 119 podocytes from left to right). Asterisks in the images indicate WT1+ podocytes. Scale bars: 10 μm and 5 μm (enlarged insets). ***P < 0.001, by 2-tailed Student’s t test. (D) Growth curves of male and female WT Sel1Lfl/fl, heterozygous Sel1LPodCre/+, and knockout Sel1LPodCre mice. Ten-week-old Ire1αPodCre mice were included as a control. *P < 0.05 and ***P < 0.001, by 1-way ANOVA for each age. (E–G) Kaplan-Meier survival analysis for combined (E), male (F), and female (G) sexes. ***P < 0.0001, by log-rank test comparing Sel1LPodCre mice with other cohorts. Values represent the mean ± SEM.

Article Snippet: The following antibodies were used for immunofluorescence of human samples: SEL1L ( 43 ) (home-made {"type":"entrez-nucleotide","attrs":{"text":"E12049","term_id":"22027566","term_text":"E12049"}} E12049 , specific for both human and mouse, rabbit, 1:500); HRD1 (provided by Richard Wojcikiewicz, SUNY Upstate Medical University, Syracuse, New York, USA; rabbit, 1:50 for immunostaining); and WT1–Alexa Fluor 647 (Abcam, ab202639, 1:500). scRNA-Seq of the human kidney.

Techniques: Quantitation Assay, Knock-Out, Control

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 for immunofluorescence of human samples: SEL1L ( 43 ) (home-made {"type":"entrez-nucleotide","attrs":{"text":"E12049","term_id":"22027566","term_text":"E12049"}} E12049 , specific for both human and mouse, rabbit, 1:500); HRD1 (provided by Richard Wojcikiewicz, SUNY Upstate Medical University, Syracuse, New York, USA; rabbit, 1:50 for immunostaining); and WT1–Alexa Fluor 647 (Abcam, ab202639, 1:500). scRNA-Seq of the human kidney.

Techniques: Western Blot, Immunoprecipitation, Control, Staining, Transfection, Quantitation Assay, Ubiquitin Proteomics

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 of WT and mutant HMW and monomeric nephrin in transfected WT and HRD1–/– HEK293T cells under nonreducing and reducing conditions. (B and C) Western blot analysis of NP40-soluble (B) and NP40-insoluble fractions (C) in transfected WT and HRD1–/– HEK293T cells, showing increased formation of HMW and insoluble nephrin aggregates in HRD1–/– HEK293T cells for both WT and mutant nephrin. (D) Western blot analysis of nephrin HMW aggregation in HEK293T cells transfected with different combinations of Myc-WT nephrin and nephrin mutants under nonreducing conditions, with the level of HMW nephrin from 1 representative experiment shown below the blot. (E and F) Western blot analysis of Myc-WT and Flag-mutant nephrin in HEK293T cells transfected with different combinations of Myc-WT nephrin and Flag-tagged mutant nephrin at a 1:1 or 1:3 ratio. Quantitation of the percentage of a form WT nephrin in total WT nephrin is shown in F, indicating a decrease in the percentage of a form WT nephrin in HRD1–/– HEK293T cells (upon cotransfection of an increased amount of mutant nephrin) when compared with that in WT HEK293T cells. Values represent the mean ± SEM. Data are representative of at least 2 independent experiments.

Article Snippet: The following antibodies were used for immunofluorescence of human samples: SEL1L ( 43 ) (home-made {"type":"entrez-nucleotide","attrs":{"text":"E12049","term_id":"22027566","term_text":"E12049"}} E12049 , specific for both human and mouse, rabbit, 1:500); HRD1 (provided by Richard Wojcikiewicz, SUNY Upstate Medical University, Syracuse, New York, USA; rabbit, 1:50 for immunostaining); and WT1–Alexa Fluor 647 (Abcam, ab202639, 1:500). scRNA-Seq of the human kidney.

Techniques: Western Blot, Mutagenesis, Transfection, Quantitation Assay, Cotransfection