Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Heparan Sulfate, Including that in Bruch's Membrane, Inhibits the Complement Alternative Pathway: Implications for Age-related Macular Degeneration

doi: 10.4049/jimmunol.0903596

Figure Lengend Snippet: CFH binding to Bruch’s membrane/choroid. Immunoblot of CFH variant binding to a single 6 mm Bruch’s membrane/choroid punches isolated from different regions of the human eye. Peripheral (P), central (C) or macula (M) punches were incubated with buffer alone (O) or in buffer with V62/Y402 (VY) or V62/H402 (VH) variants of CFH. Following incubation the punches were washed thoroughly and added to 35 μl of XT sample buffer. The sample was vortexed vigorously for 30 seconds, left at room temperature for 10 min, vortexed again, spun down and supernatant was run on a gel as previously described. The gel was transferred to nitrocellulose and probed with mouse monoclonal anti-human CFH (Serotec, OX24). Immunoreactive proteins were visualized with ECL and quantified by densitometry, n=18 for both VH and VY on peripheral punches, n=13 for both on central punches and n=3 for macular punches. (B) No statistical difference in the amount of each added variant of CFH bound to the Bruch’s membrane/choroid tissue was detected (p> 0.07), no matter where the punches were taken from. Very little endogenous CFH is detected in the punches with no added CFH.

Article Snippet: 96-well Nunc Maxisorp plates (eBioscience) were coated with 5 μg anti-CFH IgG fraction in 200 μl coating buffer (0.1 M sodium bicarbonate, PH 9.5) overnight at 4°C and blocked with 1% BSA/PBS for 1 h. Wells were washed 3 times with 0.05% Tween-20/PBS and incubated with diluted CFH samples for 1 h. After washes, monoclonal mouse anti-CFH antibody (OX 24, 1:500) (Serotec) was added for 1 h followed by a HRP-conjugated donkey anti-mouse secondary antibody (1:10000) (Jackson ImmunoResearch Laboratories) for 1 h. CFH was detected by using o-Phenylenediamine (OPD) substrate (Sigma).

Techniques: Binding Assay, Membrane, Western Blot, Variant Assay, Isolation, Incubation

Journal: bioRxiv

Article Title: Proteogenomic analysis unveils the HLA Class I presented immunopeptidome in melanoma and EGFR mutant lung adenocarcinoma

doi: 10.1101/2020.08.04.236331

Figure Lengend Snippet: a) Overview of proteogenomic analysis pipeline for HLA immunopeptidome using MS-based proteomics and next generation sequencing (NGS). HLA Class I peptides were purified from two melanoma primary cell lines, two lung adenocarcinoma cell lines and a lung adenocarcinoma tumor and analyzed by high-resolution tandem MS. b) PC9 HLA Class I-associated peptide log2 transformed intensity identified by MS, and the Pearson’s correlation coefficients show high correlation among three biological replicates. c) Total number of peptides identified in the Class I immunopeptidomes from patient-derived melanoma and EGFR mutant lung cancer cells lines and tumor. d) The peptide length distribution within the Class I immunopeptidome from all samples shows that 9mer peptides are the most abundant. e) IceLogo motif analysis of four monoallelic-restricted 9mer peptidomes (HLA-A*02, A*24, B*39 and C*07) retrieved from experimentally validated IEDB database (upper panel) and MS-identified 9mer peptidome (lower panel) in PC9 cells. Percent difference stands for the % difference in frequency of the amino acids at a location f) NetHLApan 4.0 prediction algorithm-based scoring of each MS-identified peptide and distribution of binding scores among 8-14mer peptides. Upper panel shows the distribution of total identified peptides, binders (%Rank<2.0) and strong binders (%Rank<.5). Lower panel shows box plots of the lowest predicted %Rank for the corresponding HLA Class I allele for each peptide length. g) Number of predicted binders (%Rank<2.0) assigned to different HLA alleles for each sample based on Seq2HLA typing results.

Article Snippet: Subsequently, separated proteins were transferred from gel to polyvinylidene fluoride membrane and incubated with primary anti-HLA Class I mouse HRP mono-antibody, at 1;5000, (EMR8-5, Funakoshi) overnight at 4C, then briefly incubated with SuperSignal HRP substrates (Thermo Scientific) before imaging.

Techniques: Next-Generation Sequencing, Purification, Transformation Assay, Derivative Assay, Mutagenesis, Binding Assay

Journal: bioRxiv

Article Title: Proteogenomic analysis unveils the HLA Class I presented immunopeptidome in melanoma and EGFR mutant lung adenocarcinoma

doi: 10.1101/2020.08.04.236331

Figure Lengend Snippet: a) Detailed strategic workflow of MS raw file processing, database search, cancer antigen-derived peptides identification and validation. b) Number of somatic mutations identified from NCI patient melanoma tumor derived cell lines (NCI-3784Mel and NCI-3795Mel) and an EGFR mutant lung adenocarcinoma tumor, procured at autopsy, from a patient treated with osimertinib (NCI-RA007). c) Immunoblots of total HLA Class I protein expression using pan HLA Class I (antibody clone W/32). d) Flow cytometry analysis of cell surface HLA Class I protein expression.

Article Snippet: Subsequently, separated proteins were transferred from gel to polyvinylidene fluoride membrane and incubated with primary anti-HLA Class I mouse HRP mono-antibody, at 1;5000, (EMR8-5, Funakoshi) overnight at 4C, then briefly incubated with SuperSignal HRP substrates (Thermo Scientific) before imaging.

Techniques: Derivative Assay, Mutagenesis, Western Blot, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: Proteogenomic analysis unveils the HLA Class I presented immunopeptidome in melanoma and EGFR mutant lung adenocarcinoma

doi: 10.1101/2020.08.04.236331

Figure Lengend Snippet: a) Subcellular localization and b) molecular function classification of total identified HLA Class I-associated immunopeptides using Ingenuity Pathway Analysis. c) Proteomap analysis ( www.proteomaps.net ) of total identified HLA Class I-associated peptide source proteins, considering the annotated protein molecular function and abundance. d) Canonical pathways enriched among the identified HLA Class I-associated immunopeptide parent proteins which are grouped into either lung adenocarcinoma (i.e., PC9, H1975, NCI-RA007) and melanoma (i.e., NCI-3784mel and NCI-3795Mel) or cell lines (i.e., PC9, H1975, NCI-3784mel and NCI-3795Mel) and tumor tissue (i.e, NCI-RA007). e) Oncogenes and tumor suppressors, predicted by upstream regulator analysis, to be key regulators of the HLA immunopeptide source proteins. f) Network analysis of the predicted key upstream regulators of the of Class I peptide source proteins. g) Key oncogene and tumor suppressor Class I peptides identified in this study. Peptides have not reported to date as Class I presented are indicated with *.

Article Snippet: Subsequently, separated proteins were transferred from gel to polyvinylidene fluoride membrane and incubated with primary anti-HLA Class I mouse HRP mono-antibody, at 1;5000, (EMR8-5, Funakoshi) overnight at 4C, then briefly incubated with SuperSignal HRP substrates (Thermo Scientific) before imaging.

Techniques:

Journal: bioRxiv

Article Title: Proteogenomic analysis unveils the HLA Class I presented immunopeptidome in melanoma and EGFR mutant lung adenocarcinoma

doi: 10.1101/2020.08.04.236331

Figure Lengend Snippet: a) Workflow of integrated proteogenomic analysis using germline (PBMCs) and tumor cell line/tissue WES and RNAseq datasets to identify SNVs, INDELs and fusions and construction of tumor cell line/tumor tissue-specific databases to interrogate the MS data of Class I -associated peptides. b) List of 12 mutated neopeptides, with its variant, predicted HLA-allele restriction, dbSNP ID and synthetic peptide validation and de novo sequencing search status. c-d) Box plots show peptide intensity of wildtype and mutant nonpeptides in all biological replicates from (c) H1975 and (d) PC9. e-f) Matched MS2 spectra of endogenous and its synthetic counterpart for (e) RIF1-G836S-derived neopeptide SITSIISSV and (f) ULK1p.T816A-derived neopeptide FADPIAANL. g) T2 cell-based HLA stability assay shows that RIF1 p.G836S -derived peptide (SIT S IISSV) bound and stabilized HLA-A*02. h) Box plot shows statistically significant increase of HLA expression (log2 geometric mean of counts) in T2 cells incubated with RIF1 p.G836S -derived peptide (SIT S IISSV) and positive control NY-ESO1-derived peptide compared to those incubated with DMSO (p<0.005).

Article Snippet: Subsequently, separated proteins were transferred from gel to polyvinylidene fluoride membrane and incubated with primary anti-HLA Class I mouse HRP mono-antibody, at 1;5000, (EMR8-5, Funakoshi) overnight at 4C, then briefly incubated with SuperSignal HRP substrates (Thermo Scientific) before imaging.

Techniques: Variant Assay, Sequencing, Mutagenesis, Derivative Assay, Stability Assay, Expressing, Incubation, Positive Control

Journal: bioRxiv

Article Title: Proteogenomic analysis unveils the HLA Class I presented immunopeptidome in melanoma and EGFR mutant lung adenocarcinoma

doi: 10.1101/2020.08.04.236331

Figure Lengend Snippet: a-b) Fraction of Class I -associated peptides predicted to be HLA binders (NetMHCpan %rank<2.0) or non-binders (%Rank>2.0) by (a) database search and (b) de novo sequencing. c) Correlation of peptide intensities from de novo sequencing algorithm-searched peptides from two PC9 biological replicates. d) Distribution of total number of de novo sequencing-searched 8-14mer peptides, HLA binders (%Rank<2.0) and strong binders (%Rank<0.5) (upper panel). Distribution of HLA binding affinity (%Rank) of d e novo sequencing-searched 8-14mer peptides (lower panel). e) Number of d e novo sequencing-searched binders assigned to different HLA alleles. f-j) Comparison of 9 mer peptide binding motifs (binders only, NetMHCPan %Rank<2.0) identified by database search (DB) search versus d e novo search in (f) NCI-3784Mel, (g) NCI-3795Mel, (h) PC9, (i) H1975 and (j) NCI-RA007. k) Of 12 mutated neopeptides identified by cell line/tumor specific DB search, six were also identified by de novo search. L) Matched MS2 spectra of one representative endogenous neopeptide, EIF3B p.S64P (AEAGPE P EV), identified by de novo search (upper panel), proteogenomic DB search (middle panel) and direct injection of its synthetic peptide (lower panel).

Article Snippet: Subsequently, separated proteins were transferred from gel to polyvinylidene fluoride membrane and incubated with primary anti-HLA Class I mouse HRP mono-antibody, at 1;5000, (EMR8-5, Funakoshi) overnight at 4C, then briefly incubated with SuperSignal HRP substrates (Thermo Scientific) before imaging.

Techniques: Sequencing, Binding Assay, Injection

Journal: bioRxiv

Article Title: Proteogenomic analysis unveils the HLA Class I presented immunopeptidome in melanoma and EGFR mutant lung adenocarcinoma

doi: 10.1101/2020.08.04.236331

Figure Lengend Snippet: a) Workflow used to identify lncRNA-derived peptides enriched from cancer cells and tumors. We query the de novo sequencing-searched Class I-associated peptide pool against a database generated using six-frame translated lncRNAs compiled in LNCipedia database (right workflow). The statistical significance of our algorithm was determined by potential matching of the de novo -searched peptides against a “mock” database created by the randomly picked gene blocks (∼50,000 transcripts) from hg38, which resulted in an empirical p-value<1.0e -5 (left workflow). b) 44 lncRNA-derived peptides identified using our algorithm with their predicted HLA alleles and binding affinity. c) Log2 peptide intensities of database (DB) searched, de novo searched and lncRNA-derived peptides. d) The classification of source lncRNAs for the identified lncRNA-derived peptides into antisense, sense intronic and classic lncRNAs. e) LncRNA-derived peptides that match new open reading frame (ORF), introns of coding genes, and noncoding region. f) The top panel displayed a snapshot of IGV showing lncRNA PVT1-derived peptide FLLSSSLTL, identified in PC9, with chromosomal location alignment of RNA-seq of all 5 samples and peptide BLAT; the middle panel shows the ribo-seq searching results from GWIPS; the lower panel shows the predicted binding affinity of this peptide to HLA-A*02:06 that is expressed only in PC9 cells. g and h) Matched MS2 spectra of in vivo and synthetic lncRNA-derived peptides YSFPELTHL (g) and MEHVSPALP (h) , respectively. i) T2 cell-based HLA stability assay of three lncRNA peptides predicted to be HLA-A*02 binders, FLLSSSLTL, QEEAALKAL and SLHASLSTV, and the NY-ESO-1-dervied positive control peptide.

Article Snippet: Subsequently, separated proteins were transferred from gel to polyvinylidene fluoride membrane and incubated with primary anti-HLA Class I mouse HRP mono-antibody, at 1;5000, (EMR8-5, Funakoshi) overnight at 4C, then briefly incubated with SuperSignal HRP substrates (Thermo Scientific) before imaging.

Techniques: Derivative Assay, Sequencing, Generated, Binding Assay, RNA Sequencing Assay, In Vivo, Stability Assay, Positive Control

Journal: Journal of Virology

Article Title: Kaposi's Sarcoma-Associated Herpesvirus-Positive Primary Effusion Lymphoma Tumor Formation in NOD/SCID Mice Is Inhibited by Neomycin and Neamine Blocking Angiogenin's Nuclear Translocation

doi: 10.1128/JVI.01920-13

Figure Lengend Snippet: Expression of angiogenin in Kaposi's sarcoma and PEL human samples. (A) Angiogenin expression in Kaposi's sarcoma samples. Sections of normal skin or KS tumors were analyzed by immunofluorescence staining for ANG (green) and LANA-1 (red) and counterstained with DAPI (blue). Arrows indicate colocalization of ANG and LANA-1 in KS lesions. (B and C) Angiogenin expression in lung PEL human samples. Sections of normal lung and PEL solid lung metastasis were analyzed by immunofluorescence staining for ANG (green) and the B-lymphocyte antigen CD19 (red) in panel B or LANA-1 (red) in panel C. Nuclei were visualized with DAPI staining (blue). Arrows indicate colocalization of ANG with CD19 (B) or with LANA-1 (C) in PEL lesions. Magnification, ×20.

Article Snippet: Human CD19 antibody (for WB, 1:1,000 dilution) was from GeneTex, Irvine, CA.

Techniques: Expressing, Immunofluorescence, Staining

Journal:

Article Title: Subordinate Social Status Modulates the Vulnerability to the Immunological Effects of Social Stress

doi: 10.1016/j.psyneuen.2007.09.005

Figure Lengend Snippet: Splenic response in pair housed male mice. Social status (Dominant / Submissive) was identified prior to SDR using the resident-intruder test. Pairs of mice underwent six nightly SDR cycles (SDR, n=14 pairs) or remained undisturbed in their home cages (Control, n=10 pairs). Data are presented as mean ± S.E.M. (A.) Spleen weights (gr); (B.) Splenocyte numbers; (C.) Cell viability (optical density, OD) of LPS-stimulated splenocytes treated with corticosterone (0-5 μM); (D.) TNF-α and (E.) IL-6 levels (pg/ml) secreted from LPS-stimulated splenocytes treated with corticosterone (0-5 μM); * Significantly different from all other groups (p<0.05).

Article Snippet: Tumor Necrosis Factor-α concentrations were measured by an OptEIASet Mouse TNF-α (Mono/Poly) ELISA from Pharmingen (San Diego, CA) following the manufacturer's instructions with minor modifications ( Avitsur et al., 2003a ).

Techniques:

Journal:

Article Title: Subordinate Social Status Modulates the Vulnerability to the Immunological Effects of Social Stress

doi: 10.1016/j.psyneuen.2007.09.005

Figure Lengend Snippet: Splenic response in pair housed male mice. Social status (Dominant / Submissive) was identified after SDR using the resident-intruder test. Pairs of mice underwent six nightly SDR cycles (SDR, n=14 pairs) or remained undisturbed in their home cages (Control, n=10 pairs). Data are presented as mean ± S.E.M. (A.) Spleen weights (gr); (B.) Splenocyte numbers; (C.) Cell viability (optical density, OD) of LPS-stimulated splenocytes treated with corticosterone (0-5 μM); (D.) TNF-α and (E.) IL-6 levels (pg/ml) secreted from LPS-stimulated splenocytes treated with corticosterone (0-5 μM); * Significantly different from all other groups (p<0.05).

Article Snippet: Tumor Necrosis Factor-α concentrations were measured by an OptEIASet Mouse TNF-α (Mono/Poly) ELISA from Pharmingen (San Diego, CA) following the manufacturer's instructions with minor modifications ( Avitsur et al., 2003a ).

Techniques:

Journal:

Article Title: Subordinate Social Status Modulates the Vulnerability to the Immunological Effects of Social Stress

doi: 10.1016/j.psyneuen.2007.09.005

Figure Lengend Snippet: Splenic response in pair housed male mice in cages with stable social status. Social status (Dominant / Submissive) was identified before and after SDR using the resident-intruder test. Only cages with identical social order before and after stress were included in this analysis. Pairs of mice underwent six nightly SDR cycles (SDR, n=8 pairs) or remained undisturbed in their home cages (Control, n=9 pairs). Data are presented as mean ± S.E.M. (A.) Spleen weights (gr); (B.) Splenocyte numbers; (C.) Cell viability (optical density, OD) of LPS-stimulated splenocytes treated with corticosterone (0-5 μM); (D.) TNF-α and (E.) IL-6 levels (pg/ml) secreted from LPS-stimulated splenocytes treated with corticosterone (0-5 μM);* Significantly different from all other groups (p<0.05). a Significantly different from SDR-dominant group (p<0.05).

Article Snippet: Tumor Necrosis Factor-α concentrations were measured by an OptEIASet Mouse TNF-α (Mono/Poly) ELISA from Pharmingen (San Diego, CA) following the manufacturer's instructions with minor modifications ( Avitsur et al., 2003a ).

Techniques:

Journal: Developmental Cell

Article Title: CCPG1 Is a Non-canonical Autophagy Cargo Receptor Essential for ER-Phagy and Pancreatic ER Proteostasis

doi: 10.1016/j.devcel.2017.11.024

Figure Lengend Snippet:

Article Snippet: Mouse mono anti-Syntaxin17 (clone 2F8) – IF , MBL , M212-3.

Techniques: Affinity Purification, Peptide Microarray, Control, Virus, Recombinant, Protease Inhibitor, Mass Spectrometry, Polymer, SYBR Green Assay, Stable Transfection, Expressing, Plasmid Preparation, Software, Microscopy

Journal: Nutrients

Article Title: Effect of a Semi-Purified Oligosaccharide-Enriched Fraction from Caprine Milk on Barrier Integrity and Mucin Production of Co-Culture Models of the Small and Large Intestinal Epithelium

doi: 10.3390/nu8050267

Figure Lengend Snippet: Relative fold change of MUC mRNA from epithelial cell cultures incubated with 4 mg/mL caprine milk oligosaccharide-enriched fraction (CMOF). The expression of mucin genes MUC4 , MUC2 and MUC5AC by 100:0, 90:10, 75:25, and 0:100 Caco-2:HT29-MTX cell cultures after 12 h incubation with 4.0 mg/mL CMOF, compared to respective control monolayers. The data are expressed as the mean fold change (±SEM) of three replicates across three independent experiments. A statistically significant difference in fold change is indicated by * ( p < 0.05).

Article Snippet: The blocking agent was decanted and 50 μL of either MUC2 mouse mono-clonal antibody (clone 4A4, 1:250; Creative Biomart, New York, NY, USA), MUC4 mouse mono-clonal antibody (clone 5B12, 1:500; Abnova, Taipei, Taiwan) or MUC5AC mouse mono-clonal antibody (clone 2H7, 1: 250; Abnova) in 1% BSA was added to wells and plates incubated at room temperature for 1 h. Primary antibody was decanted and all plates were washed once with 0.1% PBS-Tween (Tween 20; Sigma-Aldrich) followed by an additional three washes with PBS.

Techniques: Incubation, Expressing, Control

Journal: Nutrients

Article Title: Effect of a Semi-Purified Oligosaccharide-Enriched Fraction from Caprine Milk on Barrier Integrity and Mucin Production of Co-Culture Models of the Small and Large Intestinal Epithelium

doi: 10.3390/nu8050267

Figure Lengend Snippet: The abundance of the individual mucin proteins (MUC4, MUC2 and MUC5AC) and the total mucin protein abundance from post-confluent (21 days post-seeding) control Caco-2:HT29-MTX mono- and co-cultures (ng/µg total protein) and those incubated with 4.0 mg/mL caprine milk oligosaccharide-enriched fraction (CMOF) for 12 h. Results are expressed as the mean abundance (ng/µg of total protein) (±SEM). * p < 0.05 compared to respective control.

Article Snippet: The blocking agent was decanted and 50 μL of either MUC2 mouse mono-clonal antibody (clone 4A4, 1:250; Creative Biomart, New York, NY, USA), MUC4 mouse mono-clonal antibody (clone 5B12, 1:500; Abnova, Taipei, Taiwan) or MUC5AC mouse mono-clonal antibody (clone 2H7, 1: 250; Abnova) in 1% BSA was added to wells and plates incubated at room temperature for 1 h. Primary antibody was decanted and all plates were washed once with 0.1% PBS-Tween (Tween 20; Sigma-Aldrich) followed by an additional three washes with PBS.

Techniques: Quantitative Proteomics, Control, Incubation