total ire1  (Cell Signaling Technology Inc)

Journal: Viruses

Article Title: Induction of the Unfolded Protein Response during Bovine Alphaherpesvirus 1 Infection

doi: 10.3390/v12090974

Figure Lengend Snippet: BoHV-1 induces UPR through all three signaling branches. ( A ) Immunoblot analysis of ATF6-FL, ATF6-N, total PERK, eIF2α, and IRE1, phosphorylated IRE1, PERK, and eIF2α protein levels in MDBK cells after 12 and 24 h of BoHV-1 infection at an MOI of 0.1. ( B , C , E , F ) qPCR analysis of ATF6, GRP94, ERdj4, and ATF4 mRNA expression in MDBK cells after 12 and 24 h of BoHV-1 infection (0.1 MOI). The graph shows the levels of the genes normalized against β-actin and the data are shown as fold changes and were normalized to control cell data. ( D ) XBP1 splicing assay. First, MDBK cells were infected with BoHV-1 at 0.1 MOI at 12 or 24 hpi and harvested for RT-PCR analysis of XBP1 mRNA splicing. The production of XBP1 was digested with the restriction enzyme Pst I, and then the digested PCR products were separated by 1.5% agarose gel electrophoresis. The mean ± SD of data from three independent experiments are shown; * p < 0.05, ** p < 0.01.

Article Snippet: Antibodies against total IRE1 (3294S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Infection, Expressing, Control, Splicing Assay, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis

Journal: Viruses

Article Title: Induction of the Unfolded Protein Response during Bovine Alphaherpesvirus 1 Infection

doi: 10.3390/v12090974

Figure Lengend Snippet: The IRE1 pathway induced by BoHV-1 infection promotes viral replication in MDBK cells. ( A ) The silencing efficiency of IRE1 was measured by qPCR in MDBK cells at 24 h post-transfection. ( B ) The CCK-8 assays were used to analyze the viability of MDBK cells 24 h after transfection. ( C ) Immunoblot analysis of the silencing efficiency of IRE1 at 48 h post-transfection. ( D ) At 48 h siRNA duplexes post transfection, MDBK cells were challenged with BoHV-1 (0.1 MOI) and then harvested at 24 hpi for XBP1 splicing assay. ( E , F ) At 48 h siCon or siIRE1 post transfection, MDBK cells were infected with BoHV-1 (0.1 MOI) and harvested at the indicated time points for virus titration analyses and viral gene expression analyses. The mean ± SD of data from three independent experiments are shown; * p < 0.05, ** p < 0.01.

Article Snippet: Antibodies against total IRE1 (3294S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Infection, Transfection, CCK-8 Assay, Western Blot, Splicing Assay, Virus, Titration, Gene Expression

Journal: Viruses

Article Title: Induction of the Unfolded Protein Response during Bovine Alphaherpesvirus 1 Infection

doi: 10.3390/v12090974

Figure Lengend Snippet: BoHV-1 replication is enhanced by inhibiting IRE1 RNase activity in MDBK cells. ( A ) The efficiency of 4μ8C in inhibiting the IRE1 RNase activity was evaluated by detecting the ERdj4 mRNA levels in MDBK cells. The cytotoxicity of 4μ8C was measured using a CCK-8 assay. ( B – E ) MDBK cells were treated with 4μ8C (100 µM) or DMSO, followed by infection with BoHV-1 (0.1 MOI). ( B ) MDBK cells were harvested at 24 hpi and subjected to XBP1 splicing assay. ( C ) MDBK cells were harvested at 0, 12, and 24 hpi, and qPCR analysis of ERdj4 mRNA levels. ( D ) MDBK cells were harvested at 12 and 24 hpi and viral titers of BoHV-1 were determined. ( E ) qPCR analysis of the BoHV-1 gC expression in MDBK cells at 12 and 24 hpi. ( F ) Effect of IRE1 RNase activity inhibition on BoHV-1 replication stages. The mean ± SD of data from three independent experiments are shown; * p < 0.05, ** p < 0.01.

Article Snippet: Antibodies against total IRE1 (3294S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activity Assay, CCK-8 Assay, Infection, Splicing Assay, Expressing, Inhibition

Journal: Viruses

Article Title: Induction of the Unfolded Protein Response during Bovine Alphaherpesvirus 1 Infection

doi: 10.3390/v12090974

Figure Lengend Snippet: Inhibition of IRE1 kinase activity and promotion of RNase activity decrease BoHV-1 replication. ( A ) The cytotoxicity of APY29 was measured using a CCK-8 assay. ( B ) MDBK cells were treated with different concentrations of APY29, and then challenged with BoHV-1. Immunoblot analysis of total IRE1 and phosphorylated IRE1 in MDBK cells at 24 hpi. ( C ) MDBK cells were treated with APY29 (4 µM) or DMSO, and followed by infection with BoHV-1. Cells were harvested at 24 hpi and subjected to XBP1 splicing assay. ( D – F ) MDBK cells treated with APY29 (4 μM) or DMSO were challenged with BoHV-1 (0.1 MOI). Cells were harvested at 12 and 24 hpi. ( D ) Immunoblot analysis of the IRE1 kinase activity at the indicated time points. ( E ) The viral titers of BoHV-1 in MDBK cells at 12 and 24 hpi were determined. ( F ) qPCR analysis of the BoHV-1 gC expression in MDBK cells at the indicated time points. ( G ) Effect of IRE1 kinase activity inhibition and RNase activity enhancement on BoHV-1 replication stages. The mean ± SD of data from three independent experiments are shown; * p < 0.05, ** p < 0.01.

Article Snippet: Antibodies against total IRE1 (3294S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Inhibition, Activity Assay, CCK-8 Assay, Western Blot, Infection, Splicing Assay, Expressing

Journal: Life Science Alliance

Article Title: Paneth cell α-defensin misfolding correlates with dysbiosis and ileitis in Crohn’s disease model mice

doi: 10.26508/lsa.201900592

Figure Lengend Snippet: (A, B) Representative transmission electron microscopy images of Paneth cells at the base of ileal crypts in (A) ICR and (B) SAMP1/YitFc mice. Scale bars indicate 2 μm. (C, D) Quantitative analysis of (C) granule number and (D) ER lumen diameter in Paneth cells (n = 3/each week for SAMP1/YitFc mice). For the measurements, three Paneth cells were randomly selected from each mouse. (E) SDS–PAGE Western blot analysis of ER stress markers, pIRE1α, ATF4, cleaved-ATF6, and GRP78 in ileal crypts (n = 4/each group). Total-IRE1α and HPRT1 was used as loading control. (F) Relative expression level of ER stress markers calculated from the band intensity. Error bars represent mean ± SEM. (C, D, F) Statistical significance was evaluated by t test in (C, D), and one-way ANOVA followed by Tukey’s post hoc test in (F). P < 0.05 was considered statistically significant. * P < 0.05, † P < 0.01, § P < 0.001. E, ER; G, granules; N, nucleus; n.s., not significant.

Article Snippet: After transfer, the membranes were blocked by 5% BSA (Sigma-Aldrich) in 0.1% TBS-T at 4°C for overnight and then reacted with the following primary antibodies: anti-ATF4 (1/1,000, #11815; Cell Signaling Technology), anti-phospho-IRE1α (1 μg/ml, NB100-2323; Novus Biologicals), anti-total IRE1α (1 μg/ml, NB100-2324; Novus Biologicals), anti-ATF6 (1 μg/ml, NBP1-40256; Novus Biologicals), anti-GRP78 (1/1,000, #3177; Cell Signaling Technology), and anti-HPRT1 (0.018 μg/ml, ab109021; Abcam) at 4°C overnight.

Techniques: Transmission Assay, Electron Microscopy, SDS Page, Western Blot, Control, Expressing