Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay

Journal:

Article Title: Convergence of Heat Shock Protein 90 with Ubiquitin in Filamentous ?-Synuclein Inclusions of ?-Synucleinopathies

doi: 10.2353/ajpath.2006.050770

Figure Lengend Snippet: List of Antibodies Used in This Study

Article Snippet: 45 Triple-labeling FIHC studies were performed by co-incubating sections with antibodies specific for Hsp90, α-syn, and tau or ubiquitin, raised in different species, followed by visualizing bound antibodies with secondary antibodies conjugated with Alexa Fluor 488, Texas Red, or AMCA (Vector Laboratories) as described.

Techniques:

Journal:

Article Title: Convergence of Heat Shock Protein 90 with Ubiquitin in Filamentous ?-Synuclein Inclusions of ?-Synucleinopathies

doi: 10.2353/ajpath.2006.050770

Figure Lengend Snippet: Screening of Hsps using double-label FIHC with different anti-Hsp and anti-α-syn antibodies visualized by Alexa Fluor 594 or Texas Red and Alexa Fluor 488, respectively, shows co-localization of a subset of Hsps examined in LBs (a–g) and GCIs (h–n). Yellow color represents co-localization of Hsp and α-syn. Hsp27 (b), Hsp40 (c), Hsc70 (e), and Hsp90 (g) are located in LBs, whereas αB-crystallin (h) and Hsp90 (n) are located in GCIs and threads. Specificity of Hsp90 antibodies (o), 9D2 (lanes 1 and 2), and AC88 (lanes 3 and 4) is shown in HS fraction of human (lanes 1 and 3) and mouse (lanes 2 and 4) brain homogenates. Rat 9D2 does not recognize mouse Hsp90 (lane 2). Photomicrographs in p–x show modest Hsp90 IR in neurons of normal brain (p), which also is seen in α-synucleinopathy brains, in addition to more intense Hsp90 IR in LBs (arrowhead) of the PD SN (q), DLB amygdala (r), and GCIs (arrow) in the MSA pons (s). Little or no iHsp90 IR is seen in neurofibrillary tangles of AD (t, double arrowheads) and DLB (r). t: IHC profile with an antibody against Hsp90 (9D2) or tau (AT8) (inset) from adjacent sections in the CA1 region of AD hippocampus are presented for comparison. It is apparent that Hsp90 IR is reduced in neurofibrillary tangles. u: Modest Hsp90 IR is noted in ubiquitin inclusions in the hippocampus of the FTD-MND brain. Inset shows ubiquitin IR in inclusions on the adjacent section. v and w show double-label IHC with horseradish peroxidase-DAB and β-galactosidase-X-gal to illustrate co-localization of iHsp90 IR (brown) with α-syn IR (blue) in LBs (arrowhead) in the midbrain of PD (v) as well as GCIs (arrow) in the pons of MSA (w). x: No CHIP IR was found on GCI in the pons of MSA, whereas the adjacent section indicates descent α-syn IR inclusions (inset). Scale bars, 10 μm.

Article Snippet: 45 Triple-labeling FIHC studies were performed by co-incubating sections with antibodies specific for Hsp90, α-syn, and tau or ubiquitin, raised in different species, followed by visualizing bound antibodies with secondary antibodies conjugated with Alexa Fluor 488, Texas Red, or AMCA (Vector Laboratories) as described.

Techniques:

Journal:

Article Title: Convergence of Heat Shock Protein 90 with Ubiquitin in Filamentous ?-Synuclein Inclusions of ?-Synucleinopathies

doi: 10.2353/ajpath.2006.050770

Figure Lengend Snippet: Double-label FIHC confirms Hsp90 IR (Texas Red) co-localizes with α-syn IR (Alexa Fluor 488) in LBs of PD amygdala (arrowhead in a–c) and GCIs in the MSA pons (d–f). g–j: Representative images from triple-label FIHC of DLB amygdala shows differential co-localization of Hsp90 with α-syn (arrow) rather than tau-positive inclusions (arrowhead) in these preparations. Even when tau IR is located in iHsp90 IR cells, the subcellular localization of these proteins is normally discordant (asterisk). k: Semiquantitative analysis reveals the number of iHsp90 IR with α-syn- or tau IR lesions including intracytoplasmic inclusions, dystrophic neurites, and spheroids. l: iHsp90 co-localizes with α-syn to a significantly greater extent (P < 0.01) than tau in disease lesions. Asterisks indicate statistically significant difference. P < 0.05. Scale bars, 10 μm (a–j).

Article Snippet: 45 Triple-labeling FIHC studies were performed by co-incubating sections with antibodies specific for Hsp90, α-syn, and tau or ubiquitin, raised in different species, followed by visualizing bound antibodies with secondary antibodies conjugated with Alexa Fluor 488, Texas Red, or AMCA (Vector Laboratories) as described.

Techniques:

Journal:

Article Title: Convergence of Heat Shock Protein 90 with Ubiquitin in Filamentous ?-Synuclein Inclusions of ?-Synucleinopathies

doi: 10.2353/ajpath.2006.050770

Figure Lengend Snippet: Triple-label FIHC micrographs of iHsp90 (Texas Red), ubiquitin (Alexa Fluor 488), and α-syn (AMCA) of DLB amygdala (a–d) and MSA pons (e–h) indicate that iHsp90 co-localizes preferentially with ubiquitinated α-syn lesions (arrow), whereas a subset of α-syn-positive profiles are ubiquitin-negative (arrowhead). i and j: Data from semiquantitative analyses of triple-label FIHC studies of amygdala from DLB brain (n = 6 in i) and pons of MSA brains (n = 6 in j) showing that a similar percentage (∼30 to 45%) of the total number α-syn lesions (neurites plus perikaryal inclusions) are iHsp90− (α-syn + Hsp90) and ubiquitin IR (α-syn + Ubiq), whereas >90% of perikaryal α-syn inclusion lesions are iHsp90− (α-syn + Hsp90) and ubiquitin IR (α-syn + Ubiq). There were no significant differences between the number of Hsp90-positive and ubiquitin-positive α-syn inclusions. Scale bars, 10 μm (a–h).

Article Snippet: 45 Triple-labeling FIHC studies were performed by co-incubating sections with antibodies specific for Hsp90, α-syn, and tau or ubiquitin, raised in different species, followed by visualizing bound antibodies with secondary antibodies conjugated with Alexa Fluor 488, Texas Red, or AMCA (Vector Laboratories) as described.

Techniques:

Journal:

Article Title: Convergence of Heat Shock Protein 90 with Ubiquitin in Filamentous ?-Synuclein Inclusions of ?-Synucleinopathies

doi: 10.2353/ajpath.2006.050770

Figure Lengend Snippet: Representative images from Western blot analyses (WB) of HS, RIPA, and FA samples of cingulate cortex from normal (norm), PD (PD), DLB brains (DLB), and pons of MSA brain (MSA). Equal amounts of sample were analyzed by SDS-polyacrylamide gel electrophoresis. These results show that the FA fraction from PD, DLB, and MSA brains harbors accumulations of oligomeric α-syn species, while variably increased amounts of Hsp90 and Hsc70, but not Hsp70, are seen in FA fractions of all α-synucleinopathy disease brains. Additionally, some pathological accumulations of Hsp40 as well as Hsp27 and αB-crystallin were noted in PD/DLB and MSA brains. Polyubiquitinated protein species in the FA fraction were more apparent in the disease brains than normal brain.

Article Snippet: 45 Triple-labeling FIHC studies were performed by co-incubating sections with antibodies specific for Hsp90, α-syn, and tau or ubiquitin, raised in different species, followed by visualizing bound antibodies with secondary antibodies conjugated with Alexa Fluor 488, Texas Red, or AMCA (Vector Laboratories) as described.

Techniques: Western Blot, Polyacrylamide Gel Electrophoresis

Journal:

Article Title: Convergence of Heat Shock Protein 90 with Ubiquitin in Filamentous ?-Synuclein Inclusions of ?-Synucleinopathies

doi: 10.2353/ajpath.2006.050770

Figure Lengend Snippet: A: Effects of MG-132 and lactacystin (LC) on cultured cells. In oligodendrocyte primary culture (a and b), exposure to MG-132 (1 μmol/L) for 3, 5, 7, and 18 hours leads to the appearance of polyubiquitinated proteins after 18 hours, with slight up-regulation of Hsp90 levels (a). By 18 hours of incubation of different doses of MG-132 (1, 5, 10 μmol/L) or LC at one (10 μmol/L) dose there is a sharp induction of ubiquitinated proteins, accompanied by up-regulation of Hsp90 protein and mRNA level (b, bottom). Similar results are seen in OLN (c) cells transfected with tau40 plus α-syn and N2A cells (d) transfected with α-syn although the levels of tubulin and α-syn remain unchanged. Bar graphs indicate quantitation of variable Hsp90 protein levels due to MG132 treatment in respective cell types, confirming up-regulation of the protein level in all cases. B: Co-immunoprecipitation analysis using OLN-tau40-α-syn cells revealed noticeable protein-protein interactions between α-syn and Hsp90 as well as Hsc70 (bound), but not between α-syn and Hsp70 or Hsp40 (unbound). The protein complex of interest was isolated from the OLN cell homogenate using either no antibody (no Ab), α-syn Ab, Hsp90, Hsc70, Hsp70, or Hsp40 (IP Ab) and detected by SNL1 (α-syn) or AC88 (Hsp90) by Western blot (WB Ab). Input represents original material.

Article Snippet: 45 Triple-labeling FIHC studies were performed by co-incubating sections with antibodies specific for Hsp90, α-syn, and tau or ubiquitin, raised in different species, followed by visualizing bound antibodies with secondary antibodies conjugated with Alexa Fluor 488, Texas Red, or AMCA (Vector Laboratories) as described.

Techniques: Cell Culture, Incubation, Transfection, Quantitation Assay, Immunoprecipitation, Isolation, Western Blot

Journal:

Article Title: Convergence of Heat Shock Protein 90 with Ubiquitin in Filamentous ?-Synuclein Inclusions of ?-Synucleinopathies

doi: 10.2353/ajpath.2006.050770

Figure Lengend Snippet: In the brainstem of a 3-month-old control non-Tg mouse (a–c), M7 Tg mouse (data not shown), and asymptomatic α-syn (M83) Tg mouse (d–f), there are no α-syn lesions and modest Hsp90 IR. However, in older symptomatic M83 Tg mouse (9 months old), numerous α-syn inclusions are formed, which are frequently associated with iHsp90 IR (g–i, arrow) and ubiquitin IR (j–l, arrow). Western blots of non-Tg (lanes 1 to 3 in m–o) and M83Tg mouse (lanes 4 to 6 in m–o) show accumulation of α-syn (m), Hsp90, and Hsc70, but not Hsp70, Hsp40, or αB-crystallin (n) and polyubiquitinated (o) proteins (likely including α-syn) exclusively in the FA fraction (lane 6) of 9-month-old symptomatic M83 Tg mouse brain, but not in the 3-month-old non-Tg mouse brain (m–o) or 9-month-old non-Tg mouse (data not shown). Scale bars, 10 μm (a–l).

Article Snippet: 45 Triple-labeling FIHC studies were performed by co-incubating sections with antibodies specific for Hsp90, α-syn, and tau or ubiquitin, raised in different species, followed by visualizing bound antibodies with secondary antibodies conjugated with Alexa Fluor 488, Texas Red, or AMCA (Vector Laboratories) as described.

Techniques: Western Blot

Journal:

Article Title: Convergence of Heat Shock Protein 90 with Ubiquitin in Filamentous ?-Synuclein Inclusions of ?-Synucleinopathies

doi: 10.2353/ajpath.2006.050770

Figure Lengend Snippet: Double-immunoelectron microscopy reveals a close association of Hsp90 and α-syn in filamentous LBs in dopaminergic neurons of the SN in PD brain (a, c, e). α-Syn-positive (10 nm gold, arrow) filaments in the LB are also Hsp90-positive (18 nm gold, arrowhead), whereas unaffected dopaminergic neurons in the same SN section (f) reveal infrequent Hsp90 labeling (arrowhead). Image e is a high-power view of the inset in c, from the center of a LB. PG, neuronal pigment; N, nucleus.

Article Snippet: 45 Triple-labeling FIHC studies were performed by co-incubating sections with antibodies specific for Hsp90, α-syn, and tau or ubiquitin, raised in different species, followed by visualizing bound antibodies with secondary antibodies conjugated with Alexa Fluor 488, Texas Red, or AMCA (Vector Laboratories) as described.

Techniques: Immuno-Electron Microscopy, Labeling

Journal: Oncotarget

Article Title: Novel chemokine-like activities of histones in tumor metastasis

doi: 10.18632/oncotarget.11226

Figure Lengend Snippet: A-C. Histones (50 μg/ml, 24 hours) induced chemokine production and release as demonstrated with a Proteome Profiler™ Antibody Array in Hepa1-6 cells. D. Knockdown of NF-κB p65 and TLR4 (but not TLR2 and RAGE) in Hepa1-6 cells inhibited histone (50 μg/ml, 24 hours)-induced CCL9/10 release as demonstrated by ELISA assay (n=3, *, p<0.05 versus control shRNA group). E. Anti-CCL9/10 neutralizing antibody (1 mg/ml) partly inhibited histone (50 μg/ml, 24 hours)-induced Hepa1-6 cell migration (n=3, *, p<0.05).

Article Snippet: The production or release of chemokines was assayed using a Proteome ProfilerTM Antibody Chemokine Array Kit (#ARY020) from R&D Systems Inc. according to the manufacturer's instructions.

Techniques: Ab Array, Knockdown, Enzyme-linked Immunosorbent Assay, Control, shRNA, Migration

Journal: Oncotarget

Article Title: Novel chemokine-like activities of histones in tumor metastasis

doi: 10.18632/oncotarget.11226

Figure Lengend Snippet: A-B. Compared with the control group, TLR4 depletion (by using TLR4 −/− mice or TLR4 knockdown cells) or inhibition of histone release (by administration of 10 mg/kg heparin or 10 mg/kg H3 neutralizing antibody) limited the formation of lung metastasis (as shown in arrow) in mice based on tail vein injection of 3×10 6 Hepa1-6 cells (N=5 mice/group, *, p<0.05 versus control group). In contrast, control IgG (10 mg/kg) did not inhibit the formation of lung metastasis (B). C. Serum nucleosome levels were reduced after treatment with heparin in wildtype, but not in TLR4 −/− mice (N=5 mice/group, *, p<0.05 versus control group). D. Conceptual relationships between histone and tumor metastasis. Histone is a nuclear DAMP and can be released during cell injury or death. Once released, histone can promote cell migration and invasion through the TLR4-ERK-NF-κB pathway, which induces chemokine production and release.

Article Snippet: The production or release of chemokines was assayed using a Proteome ProfilerTM Antibody Chemokine Array Kit (#ARY020) from R&D Systems Inc. according to the manufacturer's instructions.

Techniques: Control, Knockdown, Inhibition, Injection, Migration

Journal: Immunity

Article Title: Signaling through the inhibitory Fc receptor FcγRIIB induces CD8 T cell apoptosis to limit T cell immunity

doi: 10.1016/j.immuni.2019.12.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Dr. Mark Cragg InVivoMAb anti-mouse CD16/32 (clone 2.4G2) BioXcell Cat# BE0307, RRID:AB_2736987 Bacterial and Virus Strains Biological Samples Emory Transplant Center Biorepository Emory IRB protocol #00046593 Chemicals, Peptides, and Recombinant Proteins GolgiPlug BD Biosciences Cat# 555029 OVA peptide 257–264 (SIINFEKL) GenScript Cat# RP10611 CTLA-4Ig (abatacept) Bristol-Myers Squibb, Abatacept (Orencia) Anti-CD28 domain antibodies Bristol-Myers Squibb Collagenase (type 1A) Sigma-Aldrich C2654 Hyaluronidase (type 1) Sigma-Aldrich H3506 Collagenase P Sigma-Aldrich Ref# 11213865001 Collagenase D Sigma-Aldrich Ref# 1088866001 Recombinant Fgl2 R&D Systems R&D Cat# 5257-FL-050 Critical Commercial Assays MACS CD8a+ T cell isolation kit, mouse Miltenyi Biotec 130-104-075 Quick-RNA MicroPrep Kit Zyma Research R1051 SMART-seq v4 cDNA synthesis kit Takara Cat# 634894 NexteraXT kit Illumina FC-131–1096 Mouse Fgl2 ELISA Biolegend Biolegend Cat# 437807 RNeasy Plus Micro Kit Qiagen Qiagen Cat# 74034 High Capacity cDNA reverse transcription kit ThermoFisher ThermoFisher Cat# 4368814 HT HG-U133 Plus PM BeadChip Affymetrix Thermo Fisher Cat# 901261 Lightning Link R-PE Antibody Labeling kit Novus Biologicals Cat # 703–0010 CountBright Beads Life Technologies Cat # C36950 Deposited Data RNAseq data NCBI Gene Expression Omnibus (GEO) GSE118439 Experimental Models: Cell Lines B16 Melanoma-OVA Brown, et al., 2001 , provided by Dr. Yang-Xin Fu Experimental Models: Organisms/Strains NCI C57Bl/6Ncr (NCI grantee program) Charles River Stock #556 NCI B6-LY5.1/Cr (NCI grantee program) Charles River Stock #564 OT-I Hogquist et al., 1994 OT-II Barnden et al., 1998 mOVA (C57Bl/6 background, H-2b) Ehst et al., 2013 Dr. Marc Jenkins B6.129P2-Aicda tm1(cre)Mnz /J ( Aicda −/− ) The Jackson Laboratory Stock #007770 B6;129S-Fcgr2btm1Ttk/J ( Fcgr2b −/− ) The Jackson Laboratory Stock #002848 EM:06078 Fcgr2b Fcgr2bB6null B6(Cg)-Fcgr2btm12Sjv/Cnbc ( Fcgr2b −/− ) European Mutant Mouse Archive; Dr. J.S.

Techniques: Control, Virus, Recombinant, Cell Isolation, cDNA Synthesis, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Antibody Labeling, Gene Expression, Mutagenesis, Software