Journal: Frontiers in cardiovascular medicine

Article Title: Pak2 Regulation of Nrf2 Serves as a Novel Signaling Nexus Linking ER Stress Response and Oxidative Stress in the Heart.

doi: 10.3389/fcvm.2022.851419

Figure Lengend Snippet: FIGURE 5 | Pak2-dependent IRE-XBP1 activation leads to enhanced Hrd1-mediated Nrf2 ubiquitination and degradation. (A) Hrd1 reporter luciferase activity was detected in Pak2 knockdown (Ad-shPak2) H9C2 cells in response to Tunicamycin in XBP1s expressing (Flag-XBP1) cardiomyocytes (n = 4). (B) The effect of active Pak2 overexpression and Hrd1 knockdown (siHrd1) (C) on Nrf2 ubiquitination was determined by HA-ubiquitin (HA-ub) immunoprecipitation and Nrf2 immunoblot (n = 3). Association of endogenous Nrf2 with Hrd1 was observed by immunoprecipitation in H9C2 cells under basal conditions (D) and in Tunicamycin stressed cells (E). The effect of tunicamycin in Hrd1 and Nrf2 binding is expressed as the change in IP/input ratio for Hrd1 and Nrf2 (n = 3). Nrf2 degradation in cardiomyocytes was assessed by cyclohexamide (CHX, 100 µg/mL) chases. Immunoblots showed that Flag-Pak2-T402E (F) and Myc-Hrd1 (G) enhance the clearance of Nrf2 in ARCMs (n = 3). (H) Immunoblots showing the effect of Keap1 knockdown (Keap1 siRNA) on Pak2-mediated Nrf2 downregulation (n = 3). Student’s t-test or 2-way ANOVA with Bonferroni correction for post-hoc comparisons were used for analyses. Data presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Two milligram of the protein extract was immunoprecipitated with antibodies against Nrf2 (Ab137550, Abcam), HA (2367S, Cell Signaling), Hrd1 (Nb100-2526, Novus), or IgG (Cell Signaling, 2729), respectively.

Techniques: Activation Assay, Ubiquitin Proteomics, Luciferase, Activity Assay, Knockdown, Expressing, Over Expression, Immunoprecipitation, Western Blot, Binding Assay

Journal: Frontiers in cardiovascular medicine

Article Title: Pak2 Regulation of Nrf2 Serves as a Novel Signaling Nexus Linking ER Stress Response and Oxidative Stress in the Heart.

doi: 10.3389/fcvm.2022.851419

Figure Lengend Snippet: FIGURE 6 | Pak2 activation alleviates ER stress-induced cell death in human cardiomyocites via Nrf2 downregulation. (A) Immunoblots and quantification of Nrf2, Hrd1 and cell death markers in tunicamycin stressed human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) infected with adenovirus expressing active Pak2 (Ad-Pak2) (n = 3). (B) Cell viability of iPSC-CMs was determined by MTT assay (n = 4). Immunoblots and quantification of Nrf2, cell death markers (C), ATR1, Angiotensinogen, and Mas1 (E) in tunicamycin stressed Pak2 knockdown iPSC-CMs under Nrf2 inhibition with ML385 (n = 3). (D) The effect on cell viability was determined by MTT assay (n = 4). (F) Immunoblots and quantification of Pak2 and Nrf2 in heart samples from healthy donors and transplantation patients with dilated heart disease (n = 5). Student’s t-test or 2-way ANOVA with Bonferroni correction for post-hoc comparisons were used for analyses. Data presented as mean ± SEM. (G) Proposed model for Pak2-mediated regulation of Nrf2 in response to ER stress. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Two milligram of the protein extract was immunoprecipitated with antibodies against Nrf2 (Ab137550, Abcam), HA (2367S, Cell Signaling), Hrd1 (Nb100-2526, Novus), or IgG (Cell Signaling, 2729), respectively.

Techniques: Activation Assay, Western Blot, Derivative Assay, Infection, Expressing, MTT Assay, Knockdown, Inhibition, Transplantation 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 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: Oncogene

Article Title: TRC8 suppresses tumorigenesis through targeting heme oxygenase-1 for ubiquitination and degradation.

doi: 10.1038/onc.2012.244

Figure Lengend Snippet: Figure 2. TRC8 promotes HO-1 ubiquitination. (a) HEK293T cells were transfected for 24 h with HA-tagged ubiquitin cDNA (HA-Ub) alone or together with His-tagged HO-1 cDNA (His-HO-1) alone or together with wild-type TRC8 cDNA (Wt-TRC8) or mutant TRC8 cDNA (Mu-TRC8) as indicated, then His-HO-1 protein was isolated by metal affinity resin and subjected to western blot analysis with antibodies against the HA tag (top panel) or HO-1 (middle panel). Expression of HO-1, TRC8 or b-actin (internal control) in whole-cell lysates (WCLs) was also examined by western blot analysis (bottom panel). (b) HEK293T cells were transfected with TRC8 cDNA alone or together with HA-Ub cDNA and cDNA encoding either His-tagged HO-1 or His-tagged truncated HO-1 lacking the C-terminal 24 residues (His-t-HO-1). After 24 h, the cells were lysed and His-tagged HO-1 or the truncated HO-1 isolated and subjected to western blot analysis with antibodies against the HA tag (top panel) or HO-1 (bottom panel). (c) HEK293T cells were transfected for 24 h with HA-Ub cDNA and His-HO-1cDNA with or without cDNA encoding TRC8, AMFR or SYVN1 as indicated, then His-HO-1 was isolated and subjected to western blot analysis as described above (top and middle panels). Expression of TRC8, AMFR and SYVN1 in the WCL was also examined by western blot analysis using specific antibodies (bottom panel).

Article Snippet: Plasmids The ubiquitin genome-wide full-length cDNA (GFC)-transfection array and plasmids containing human TRC8, AMFR or SYVN1 cDNA were obtained from OriGene (Rockville, MD, USA).

Techniques: Ubiquitin Proteomics, Transfection, Mutagenesis, Isolation, Western Blot, Expressing, Control

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