Journal: Acta Neuropathologica

Article Title: Granulovacuolar degeneration bodies are neuron-selective lysosomal structures induced by intracellular tau pathology

doi: 10.1007/s00401-019-02046-4

Figure Lengend Snippet: Overview of the primary antibodies used in the present study

Article Snippet: pIRE1α , Ser724 , Rabbit polyclonal , 1:1250 , , 1:50 , , , , Novus Biologicals , NB100-2323.

Techniques: In Vitro, Transduction

Journal: Neurobiology of disease

Article Title: The GRP78-PERK axis contributes to memory and synaptic impairments in Huntington's disease R6/1 mice.

doi: 10.1016/j.nbd.2023.106225

Figure Lengend Snippet: Fig. 3. Selective induction of PERK in the hippocampus of young R6/1 mice. Representative immunoblots showing the levels of P-PERK (Thr980) and PERK (A), fl- ATF6 (full-length) and clv-ATF6 (cleaved) (B), P-IRE1α (Ser724) and IRE1α (C) in total lysates from hippocampal extracts of WT and R6/1 male mice at 12 weeks of age. (*) indicates an unspecific band recognized by the ATF6 antibody. Actin was used as a loading control. Histograms show protein levels relative to actin and expressed as percentage of WT mice. Data represent the mean ± SEM (W, weeks; n = 6–7 mice/group). ***P < 0.001 vs WT mice determined by unpaired Student's t- test. Representative immunoblots showing the levels of P-eIF2α (Ser 51) and eIF2α (D), ATF4 (E), XBP1s (spliced form) and XBP1u (unspliced form) (F) in total lysates from hippocampal extracts of WT and R6/1 male mice at 12 weeks of age. (*) indicates an unspecific band recognized by the XBP1 antibody. Actin was used as a loading control. Histograms show protein levels relative to actin and expressed as percentage of WT mice. Data represent the mean ± SEM (W, weeks; n = 6–7 mice/group). *P < 0.05 vs WT mice determined by unpaired Student's t-test.

Article Snippet: After 1 h incubation in blocking buffer containing 10% non-fat powdered milk in TBS-T (50 mM Tris-HCl, 150 mM NaCl, pH 7.4, 0.05% Tween 20) membranes were blotted overnight (o/n) at 4 ◦C with the following primary antibodies: GRP78 (1:1000, Abcam® cat n◦ ab21685), CHOP (1:1000, Novus Biological® cat n◦ NBP2–13172), PPERK(Thr980) (1:1000, ThermoFisher® cat n◦ MA5–15033), PERK (1:1000, Cell Signaling Technology® cat n◦ 3192), P-IRE1α(Ser724) (1:1000 Novus Biological® cat n◦ NB100-2323SS), IRE1α (1:1000 Novus Biological® cat n◦ NB100-2324SS), ATF6 (1:1000 Novus Biological® cat n◦ NBP1-40256SS), eIF2α (1:1000 Cell Signaling Technology cat n◦ 9722), P-eIF2α (Ser51) (1:1000 Cell Signaling Technology cat n◦ 9721), ATF4 (1:1000 Proteintech® cat n◦ 10,835–1-AP), XBP1 (1:1000 Novus Biologicals ®cat n◦77681ss), P-PSD95(T19) (1:1000, Abcam® cat n◦ 16,496), PSD95 (1:2000, Cell Signaling Technology® cat n◦ 34,505), PCREB(Ser133) (1:1000 Millipore® cat n◦ 06–519), CREB (1:1000 Cell Signaling Technology® cat n◦ 9197S), BDNF (1:1000.

Techniques: Western Blot, Control

Journal: eLife

Article Title: Brucella activates the host RIDD pathway to subvert BLOS1-directed immune defense

doi: 10.7554/eLife.73625

Figure Lengend Snippet: ( A ) Strategy for generating Ern1 CKO (m-IRE1α) mice. ( B ) Bone marrow-derived macrophages (BMDMs) from m-IRE1α mice fail to induce Xbp1 splicing in the presence of ER stress inducer tunicamycin (Tm) at the indicated hours post treatment. wt-IRE1α: mice harboring the wild-type (WT) Ern1 . ( C ) Comparison of the levels of lymphocytes and myeloid cells in the wt-IRE1α control and m-IRE1α mice. Spleen weight of Brucella melitensis Bm16M-infected mice at 7 ( D ) or 14 ( E ) days post infection (dpi). Data represent the mean ± standard error of mean (SEM) from three independent experiments.

Article Snippet: Antibody , IRE1α antibody , Novus Biologicals; Thermo Fisher Scientific , Cat: NB100-2324; PA5-20189 , WB (1:500–1000).

Techniques: Derivative Assay, Comparison, Control, Infection

Journal: eLife

Article Title: Brucella activates the host RIDD pathway to subvert BLOS1-directed immune defense

doi: 10.7554/eLife.73625

Figure Lengend Snippet: Innate cytokine production of IL-1β ( A ), IL-6 ( B ), and TNF-α ( C ) in bone marrow-derived macrophages (BMDMs) from the wild-type (WT, wt-IRE1α) control and Ern1 conditional knockout (CKO, m-IRE1α) mice. The BMDMs were stimulated with LPS (100 ng/ml) and at 6 hr poststimulation the cytokine production of the treated cells was determined. Colony-forming unit (CFU) assay for B. melitensis 16M (Bm16M) intracellular survival in spleens and livers of wt- and m-IRE1α mice at 7 ( D ) or 14 ( E ) days post infection (dpi). ( F ) Histopathology of representative hematoxylin and eosin (H&E) stained sections of spleen and liver from Bm16M-infected wt- and m-IRE1α mice at 14 dpi. Bar: 100 μm. Quantification of inflammation of spleens ( G ) or livers ( H ) at 14 dpi. ( I ) CFU assays of CD11b + cells from Bm16M-infected wt- or m-IRE1α mice. ( J ) CFU assay for B. abortus S2308 (BaS2308) intracellular survival in spleens and livers in wt-IRE1α control or m-IRE1α mice at 7 dpi. ( K ) Bm16M invasion and intracellular replication in BMDMs from m-IRE1α and control mice. h.p.i.: hours post infection. CFU assays of Bm16M infection of WT BMDMs ( L ) or RAW264.7 macrophages ( M ). Host cells were pretreated with 4μ8C (50 μM) 1 hr before and during infection; CFUs of the infected cells were determined at the indicated h.p.i. ( N ) CFU assays for Bm16M infection of BMDMs from WT and Xbp1 knockout (Δ Xbp1 ) mice at the indicated h.p.i. CFU assay for Bm16M intracellular survival in spleen ( O ) or liver ( P ) in WT or Δ Xbp1 mice at 14 dpi. Immunoblotting assay for IRE1α expression ( Q ) and quantification of the expression levels ( R ) in BMDMs during a time course (48 hr) of Bm16M infection. Bm16M infection induces phosphorylation of host IRE1α ( S ) and quantification of the phosphorylated levels of IRE1α during a time course (24 hr) of infection ( T ). Images/blots are representative of three independent experiments. Statistical data represent the mean ± standard error of mean (SEM) from three independent experiments. *, **, and *** indicate significance at p < 0.05, 0.01, and 0.001, respectively.

Article Snippet: Antibody , IRE1α antibody , Novus Biologicals; Thermo Fisher Scientific , Cat: NB100-2324; PA5-20189 , WB (1:500–1000).

Techniques: Derivative Assay, Control, Knock-Out, Colony-forming Unit Assay, Infection, Histopathology, Staining, Western Blot, Expressing, Phospho-proteomics

Journal: eLife

Article Title: Brucella activates the host RIDD pathway to subvert BLOS1-directed immune defense

doi: 10.7554/eLife.73625

Figure Lengend Snippet: ( A ) Immunoblotting detection of IRE1α expression in the WT and Ern1 knockout (KO, Ern1 −/− ) murine embryonic fibroblasts (MEFs). Colony-forming unit (CFU) assay for Bm16M infection of WT and Ern1 −/− MEFs ( B ) and IRE1α expression in these cells during a time course (48 hr) of Bm16M infection ( C ). ( D ) The expression levels of the truncated IRE1α in m-IRE1α BMDMs during Bm16M infection. ( E ) Disruption of the endonuclease domain of IRE1α does not impair its kinase activity in response to Bm16M infection. h.p.i.: hours post infection. Images are representative of three independent experiments. Data represent the mean ± standard error of mean (SEM) from three independent experiments. ***Significance at p < 0.001.

Article Snippet: Antibody , IRE1α antibody , Novus Biologicals; Thermo Fisher Scientific , Cat: NB100-2324; PA5-20189 , WB (1:500–1000).

Techniques: Western Blot, Expressing, Knock-Out, Colony-forming Unit Assay, Infection, Disruption, Activity Assay

Journal: eLife

Article Title: Brucella activates the host RIDD pathway to subvert BLOS1-directed immune defense

doi: 10.7554/eLife.73625

Figure Lengend Snippet: Colocalization analysis of Bm16M with host early endosomes ( A ) and late endosomes ( B ) of BMDMs from wt- and m-IRE1α mice at the indicated time points postinfection. m.p.i.: minutes post infection. EEA1: early endosomal antigen 1; M6PR: mannose-6-phosphate receptor. Arrows in panel B: M6PR + -BCVs. Quantification of Bm16M entry into early endosomes ( C ) or late endosomes ( D ) of the indicated host BMDMs at the indicated time points postinfection. Colocalization analysis of Bm16M and the ER marker calreticulin ( E ), and quantification of Bm16M-calreticulin + ( F ) in wt- and m-IRE1α BMDMs at the indicated h.p.i. Colocalization of Bm16M and the lysosomal markers LAMP1 ( G ) or cathepsin D ( I ), and quantification of Bm16M-LAMP1 + ( H ) or -cathepsin D + ( J ) in wt- and m-IRE1α BMDMs at the indicated h.p.i. Images are representative of three independent experiments. Statistical data express as mean ± standard error of mean (SEM) from three independent experiments. ***p < 0.001.

Article Snippet: Antibody , IRE1α antibody , Novus Biologicals; Thermo Fisher Scientific , Cat: NB100-2324; PA5-20189 , WB (1:500–1000).

Techniques: Infection, Marker

Journal: eLife

Article Title: Brucella activates the host RIDD pathway to subvert BLOS1-directed immune defense

doi: 10.7554/eLife.73625

Figure Lengend Snippet: ( A ) Determination of IRE1α RNase activity in host cells via analysis of Xbp1 splicing. Murine J774.A1 macrophages were infected with Bm16M, Bm16MΔ virB2 , or treated with Tm (2.5 μg/ml); at the indicated h.p.i. or treatment, the infected or drug-treated cells were harvested for Xbp1 splicing analysis. Representative images from one of three independent experiments are shown. ( B ) Strategy for gene profiling via RNA-seq analysis. ( C ) Heatmap of differentially expressed genes (DEGs) in the indicated host cells infected with Bm16M at 4 and 24 h.p.i. DEGs from triplicate samples are shown. ( D ) Heatmap of endoplasmic reticulum (ER)-associated DEGs in the wt-IRE1α (WT) host cells infected (In) and uninfected (Un) with Bm16M at 24 h.p.i. (upper panel) or at 4 and 24 h.p.i. (lower panel). ( E ) Fold changes of representative DEGs.

Article Snippet: Antibody , IRE1α antibody , Novus Biologicals; Thermo Fisher Scientific , Cat: NB100-2324; PA5-20189 , WB (1:500–1000).

Techniques: Activity Assay, Infection, RNA Sequencing

Journal: eLife

Article Title: Brucella activates the host RIDD pathway to subvert BLOS1-directed immune defense

doi: 10.7554/eLife.73625

Figure Lengend Snippet: ( A ) Venn diagram showing numbers of candidate RIDD genes identified in the indicated datasets. ( B ) Common candidate RIDD genes identified in Bm16M-infected cells and other conditions in the indicated datasets. ( C ) Interaction network analysis of candidate RIDD genes ( Bloc1s1 associated genes) identified in Brucella -infected cells and the corresponding enriched KEGG pathways. Different pathways are distinguished by different colors. Interacting genes are shown with the smallest sized of dot with gene names. The upper-left-corner panel: enriched KEGG pathways and interacting candidate RIDD genes (%). qRT-PCR validation of RIDD candidate genes Bloc1s1 ( D ), Diras2 ( E ), Cd300lf ( F ), and Txnip ( G ) identified from RNA-seq analysis. Relative mRNA expression levels in potential RIDD targets from control and m-IRE1α BMDMs infected with Bm16M at 16 and 48 h.p.i. were measured by qRT-PCR. ( H ) qRT-PCR analysis of expression levels of Bloc1s1 in Δ Xbp1 BMDMs that were either uninfected (control), infected with Bm16M, or treated with tunicamycin (Tm, an UPR inducer, 5 μg/ml) at 4-hr post infection/treatment. Expression levels of the indicated genes were normalized to Gapdh expression. Statistical data represent the mean ± standard error of mean (SEM) from three independent experiments. *, **, and ***: significance at p < 0.05, 0.01, and 0.001, respectively. Figure 3—source data 1. Candidate RIDD gene identified in host cells infected by Brucella melitensis Bm16M.

Article Snippet: Antibody , IRE1α antibody , Novus Biologicals; Thermo Fisher Scientific , Cat: NB100-2324; PA5-20189 , WB (1:500–1000).

Techniques: Infection, Quantitative RT-PCR, Biomarker Discovery, RNA Sequencing, Expressing, Control

Journal: eLife

Article Title: Brucella activates the host RIDD pathway to subvert BLOS1-directed immune defense

doi: 10.7554/eLife.73625

Figure Lengend Snippet: Host cells were treated with or without the indicated drugs and at 4 hr post treatment, drug-treated or untreated host cells were fixed and subjected to confocal immunofluorescence assays. Late endosome/lysosome (LE/Lys) trafficking in m Bloc1s1 ( A ) or Rr- Bloc1s1 ( B ) and control cells in the presence of ER stress inducer tunicamycin (Tm) or IRE1α RNase inhibitor 4μ8C. The indicated cell lines were incubated with Tm, 4μ8C, or Tm + 4μ8C for 4 hr, the cells were then fixed and subjected to confocal immunofluorescence microcopy assay analysis. Bar: 10 μm. Quantification of LE/Lys retrograde trafficking in the m Bloc1s1 ( C ) or Rr- Bloc1s1 ( D ) cells treated with Tm, 4μ8C, or Tm + 4μ8C at 4 hr. U_Ctrl: untreated control. Quantification of colocalization of latex beads with Cathepsin D in m Bloc1s1 ( E ) or Rr- Bloc1s1 ( F ) cells treated with Tm, 4μ8C, or Tm +4μ8C for 4 hr. Quantification of autophagic flux (using LC3b as a marker) in m Bloc1s1 ( G ) or Rr- Bloc1s1 ( H ) cells treated with Tm, 4μ8C, or Tm + 4μ8C for 4 hr. Images are representative of three independent experiments. Statistical data represent the mean ± standard error of mean (SEM) from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: Antibody , IRE1α antibody , Novus Biologicals; Thermo Fisher Scientific , Cat: NB100-2324; PA5-20189 , WB (1:500–1000).

Techniques: Immunofluorescence, Control, Incubation, Marker

Journal: eLife

Article Title: Brucella activates the host RIDD pathway to subvert BLOS1-directed immune defense

doi: 10.7554/eLife.73625

Figure Lengend Snippet:

Article Snippet: Antibody , IRE1α antibody , Novus Biologicals; Thermo Fisher Scientific , Cat: NB100-2324; PA5-20189 , WB (1:500–1000).

Techniques: Derivative Assay, Virus, Mutagenesis, Plasmid Preparation, Cell Culture, Transfection, Construct, Control, Expressing, Recombinant, Sequencing, Synthesized, Phagocytosis Assay, Immunoprecipitation, cDNA Synthesis, Reverse Transcription, TA Cloning, Subcloning, SYBR Green Assay, Blocking Assay, Western Blot, Stripping Membranes, Molecular Weight, Staining, Software, Quantitative Proteomics, Functional Assay, Biomarker Discovery

Journal: European journal of pharmacology

Article Title: Hypothalamic pathways regulate the anorectic action of p-chloro-diphenyl diselenide in rats.

doi: 10.1016/j.ejphar.2017.09.032

Figure Lengend Snippet: Fig. 2. Effects of a single injection of (p-ClPhSe)2 (1 mg/kg; i.p.; 24 h) on protein levels of neuropeptides CART, POMC, NPY, AgRP, pro-MCH and orexin precursor (A); p-CREB/ CREB and SIRT1 (B); and ER stress-related proteins BIP, p- PERK, p-IRE1/IRE1, p-eIF2α/eIF2α, XBP-1 s and CHOP (C) in hypothalamus of non-deprived rats. HSP 90 or β-actin was used to normalize protein levels. Data analysis was carried out through (*) unpaired t-test. Significance: P < 0.05 as compared with the control group.

Article Snippet: Briefly, total protein lysates from hypothalamus (20 μg) were subjected to SDS-PAGE, electrotransferred onto a polyvinylidenedifluoride membrane, and probed with the following antibodies: BiP (GRP78) and phospho-CREB (Ser133) (p-CREB) (Cell Signaling technology, Danvers, Massachusetts); IRE1 alpha (IRE1) (Novus Biologicals); CART, Orexin-A/B (Orexin precursor), pro-MCH, p-PERK (Thr 981), GADD 153 (CHOP), CREB-1, eIF2α, HSP 90α/β (HSP 90), POMC, p-eIF2α, SIRT1 and XBP-1 (Santa Cruz Biotechnology, Santa Cruz, California); NPY, β-Actin (Sigma-Aldrich, St. Louis, MO); AGRP and IRE1 (Abcam).

Techniques: Injection, Control