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: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Sel1L is indispensable for mammalian endoplasmic reticulum-associated degradation, endoplasmic reticulum homeostasis, and survival

doi: 10.1073/pnas.1318114111

Figure Lengend Snippet: Sel1L is indispensable for the stability of Sel1L-Hrd1 complex in vivo. Pancreas was harvested at days 4, 8, and 13. (A) Western blot analysis of Sel1L and Hrd1 in WT and IKO pancreas at day 4 and 8, with quantitation shown in B upon being normalized to the loading control HSP90. (C) qPCR analysis of Sel1L and Hrd1 genes at day 13; n = 5. (D) Immunohistochemical staining of Sel1L and Hrd1 in pancreas at day 13; n = 3 each. (E) Western blot analysis of Sel1L and Hrd1 in the ileum and kidney of IKO mice. (F) Linear regression analysis between Hrd1 and Sel1L protein levels in various WT or Sel1L-deficient tissues and cell lines, including pancreas, gut, kidney, and MEFs. See SI Materials and Methods for details. Each dot represents one sample (n = 25). The slope of the regression line and the square of the correlation coefficient (R2) are shown. (G) Western blot analysis of Sel1L-associated factors (EDEM1, OS9, and XTP3B) in WT and IKO pancreas at day 13 with quantitation shown in H. (I) qPCR analysis of Edem1 and Os9 in the pancreas at day 13. (J) Percentage of Nonidet P-40 insoluble pellet weight in total tissue weight at the indicated times following tamoxifen injection; n = 2–6. WT samples were pooled from two samples of day 8 and four samples of day 13. (K) Western blot analysis of various proteins in the NP40P and NP40S fractions of pancreas at day 13 using lysis buffer containing 0.5% Nonidet P-40. The distribution of Bag6 and H2A marks the soluble (S) and insoluble (P) fractions, respectively. Representative data of three samples each shown. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by Student t test. Representative data of two experiments shown.

Article Snippet: Antibodies used for IHC were: α-Amylase (1:200), Sel1L (1:200), and K i -67 (1:50) from Abcam, and Hrd1 (rabbit, 1:200) from Novus Biologicals.

Techniques: In Vivo, Western Blot, Quantitation Assay, Control, Immunohistochemical staining, Staining, Injection, Lysis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Sel1L is indispensable for mammalian endoplasmic reticulum-associated degradation, endoplasmic reticulum homeostasis, and survival

doi: 10.1073/pnas.1318114111

Figure Lengend Snippet: Sel1L is indispensable for Hrd1 stability, cellular growth, and ER homeostasis in vitro. (A) Microscopic images of f/f;ERCre− and f/f;ERCre+ MEFs at day 4 of different passages (p). p0, vehicle (ethanol) treated; p1/2, 4-OHT treated. Quantitation of cell number from one experiment (representative of two) shown on the right. (B) Cell viability analysis as measured by CCK-8 of MEFs at day 4 of each passage. (C) Western blot analysis of Sel1L and Hrd1 in f/f;ERCre+ MEFs at day 4 of each passages. Quantitation from one representative experiment of at least three repeats shown below the gel. (D) Flow cytometric analysis of transfected A1ATNHK-GFP in p0 or p1 Sel1LIKO MEFs. GFP+ cells from each passage were gated and overlaid in the histogram (n = 3). Representative microscopic images shown on the right. Scale bar, 50 μm. (E) Xbp-1 mRNA splicing of MEFs at day 4. Quantitation of the percent of Xbp1s in total Xbp1 mRNA shown on the right. (F) Flow cytometric analysis of the ER/Golgi volume in MEFs stained with BFA-Bodipy at each passage. Quantitation of mean fluorescence intensity shown on the right. (G) Western blot analysis of eIF2α and S6 phosphorylation in MEFs at day 4 of each passage. HSP90, a loading control. Quantitation from three experiments shown on the right. (H) Polysome profiling of f/f;ERCre+ MEFs at p0 and p1. Tg (300 nM, 2 h)-treated p0 MEFs were included as a positive control for ER stress. M, monosomes; P, polysomes; P/M, ratio of polysomes to monosomes based on the quantitation of area under curve of respective fraction. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***. P < 0.01 compared with WT. Representative data of at least two experiments shown.

Article Snippet: Antibodies used for IHC were: α-Amylase (1:200), Sel1L (1:200), and K i -67 (1:50) from Abcam, and Hrd1 (rabbit, 1:200) from Novus Biologicals.

Techniques: In Vitro, Quantitation Assay, CCK-8 Assay, Western Blot, Transfection, Staining, Fluorescence, Phospho-proteomics, Control, Positive Control

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: Figure 3. Hrd1 is required for CDT intoxication. (a) Co-immunoprecipitation of Derl2 and Hrd1. Derl2 was immunoprecipitated as in figure 2i and samples were analyzed for Hrd1 by western blot. (b) CRISPR mediated deletion of Hrd1 (DHrd1) results in decreased expression as judged by western blot of Hrd1 from a-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 DHrd1 cells were intoxicated with Aa-CDT (d), Hd- CDT (e), Ec-CDT (f) and Cj-CDT (g) similar to figure 1. Percent viability is normalized to unintoxicated controls and error bars indicate standard error. (h–j) Retrograde trafficking of Hd-CDT in DHrd1 cells is blocked at the endoplasmic reticulum. pDsRed2-ER (red) transfected 293 cells and DHrd1 cells were incubated with Hd-CDT on ice, washed and incubated at 37uC for 240 minutes. Cells were then fixed and stained with DAPI (nuclei, blue) and a- Hd-CdtB (green) antibody. White scale bars indicate 5 mm. (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. doi:10.1371/journal.ppat.1004295.g003

Article Snippet: Membranes were probed with either rabbit antiDerl2 antibody (Sigma Aldrich) or rabbit anti-Hrd1 polyclonal antibody (Novus Biologicals) at a 1:2000 dilution followed by HRP conjugated a-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: Figure 6. Derl2 and Hrd1 contribute to sensitivity to Ricin, independent of the Derl2 WR motif and the interaction of Derl2 with p97. (a) Derl2 deficiency causes resistance to ricin. A745TKR cells, CHO-CDTRC1 cells, and CHO-CDTRC1 cells expressing Derl2 were seeded in a 384- well plate (16103 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 LD50 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) Derl2DC complements the resistance to ricin. CHO-CDTRC1 cells expressing empty vector, Derl2 and Derl2DC were intoxicated similar to (a). (d) The Derl2 WR motif is not required for intoxication by ricin. CHO-CDTRC1 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. doi:10.1371/journal.ppat.1004295.g006

Article Snippet: Membranes were probed with either rabbit antiDerl2 antibody (Sigma Aldrich) or rabbit anti-Hrd1 polyclonal antibody (Novus Biologicals) at a 1:2000 dilution followed by HRP conjugated a-rabbit antibody (Invitrogen) to allow detection.

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

Journal: Scientific Reports

Article Title: Acute ER stress regulates amyloid precursor protein processing through ubiquitin-dependent degradation

doi: 10.1038/srep08805

Figure Lengend Snippet: (A), CHO cells were treated with A23187 or tunicamycin for 12 h. Cells were then harvested for immunoblotting with anti-22C11 (APP), anti-USP25, anti-HRD1, anti-Bip, and anti-β-actin antibodies. (B), CHO cells pre-incubated with MG132 or vehicle for 30 min were exposed to A23187 for 12 h. Whole-cell extract of CHO cells was subjected to immunoprecipitation (IP) with anti-USP25 antibody and then immunoblotted with anti-22C11 (APP), anti-USP25, anti-HRD1, and anti-β-actin antibodies. (C), CHO cells were treated with A23187 for 12 h after transfection with USP25. The total cell lysates were analyzed by western blotting with the antibodies. For each experiment, APP level was quantified by densitometry and normalized to β-actin loading control. Statistical significance was analyzed by one-way ANOVA followed by a Tukey's multiple-comparison test. (n = 3. *P < 0.05 versus vehicle). All the gels were run under the same experimental conditions. Full-length images are presented in the .

Article Snippet: The following antibodies were used for immunodetection: anti-22C11, Nicastrin (Millipore, Billerica, MA, USA), 6E10 (Signet, Dedham, MA, USA), sAPPβ (Covance, Princeton, NJ, USA), anti-Flag (Sigma), HA (Cell Signaling Technology, Seoul, Korea), USP25 (Santa Cruz, Dallas, TX, USA), HRD1 (Abgent, Seoul, Korea), Calnexin (Enzo, Farmingdale, NY, USA), LC3B (Novus, Littleton, CO, USA), GAPDH (Abcam, Seoul, Korea), β-actin and tubulin (Sigma-Aldrich).

Techniques: Western Blot, Incubation, Immunoprecipitation, Transfection

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