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: Frontiers in Endocrinology

Article Title: A new LNC89/LNC60-Col11a2 axis revealed by whole-transcriptome analysis may be associated with goiters related to excess iodine nutrition

doi: 10.3389/fendo.2024.1407859

Figure Lengend Snippet: co-expressed mRNA of NONMMUG018089.2.

Article Snippet: Primary antibodies are rabbit anti-mouse Col11a2 antibody (YT1009, 1:500, Immunoway, China) and mouse anti-mouse β-actin antibody (TA811000, 1:2000, OriGene, USA) while mouse and rabbit second antibodies from Abbkine (A25012, 1:10000, China) and CST (14708, 1:2000, USA).

Techniques:

Journal: Frontiers in Endocrinology

Article Title: A new LNC89/LNC60-Col11a2 axis revealed by whole-transcriptome analysis may be associated with goiters related to excess iodine nutrition

doi: 10.3389/fendo.2024.1407859

Figure Lengend Snippet: Col11a2 mRNA and protein expression in goiter mouse model. (A) Col11a2 protein expression levels increased in the 10 weeks treatment group compared with the 10 weeks control group by western blotting. (B) Col11a2 protein expression levels increased in the 20 weeks treatment group compared with the 20 weeks control group by western blotting. (C) Col11a2 expression mRNA levels increased in both treatment groups detected by qRT−PCR. (D) IHC result. (D1) Quantitative analysis of IHC results. Col11a2 expression levels increased in the 10 weeks treatment 1 group compared with 10 weeks control group. Col11a2 expression levels increased in the 20 weeks treatment group compared with the 20 weeks control group. (D2) 10 weeks control group. (D3) 20 weeks control. (D4) 10 weeks treatment. (D5) 20 weeks treatment group. Scale bar = 25 μm. * P < 0.05, ** P < 0.01. *** P < 0.001.

Article Snippet: Primary antibodies are rabbit anti-mouse Col11a2 antibody (YT1009, 1:500, Immunoway, China) and mouse anti-mouse β-actin antibody (TA811000, 1:2000, OriGene, USA) while mouse and rabbit second antibodies from Abbkine (A25012, 1:10000, China) and CST (14708, 1:2000, USA).

Techniques: Expressing, Control, Western Blot, Quantitative RT-PCR

Journal: Frontiers in Endocrinology

Article Title: A new LNC89/LNC60-Col11a2 axis revealed by whole-transcriptome analysis may be associated with goiters related to excess iodine nutrition

doi: 10.3389/fendo.2024.1407859

Figure Lengend Snippet: LNC60-Col11a2 axis in Nthy-ori-1 cells. (A) After treatment with 10 5 and 5×10 6 KIO 3 for 72 h, qPCR showed that LNC60 and Col11a2 mRNA levels were significantly increased compared with those in the control group. (B) With 5×10 6 KIO 3 treated for 72 h, Wb shows Col11a2 protein level was significant increased compared with the Control group. (C) After siLNC60, qPCR showed that LNC60 and Col11a2 were significantly decreased compared with those in the NC group. (D) After siLNC60, Wb showed that the Col11a2 protein level was significantly decreased compared with that in the NC group. (E) After siLNC60, the CCK8 assay showed a significant cell viability inhibition in the 5×10 6 M KIO 3 treatment group at 72 hours. * P <0.05, ** P <0.01, *** P <0.001, t test .

Article Snippet: Primary antibodies are rabbit anti-mouse Col11a2 antibody (YT1009, 1:500, Immunoway, China) and mouse anti-mouse β-actin antibody (TA811000, 1:2000, OriGene, USA) while mouse and rabbit second antibodies from Abbkine (A25012, 1:10000, China) and CST (14708, 1:2000, USA).

Techniques: Control, CCK-8 Assay, Inhibition

Journal: Frontiers in Endocrinology

Article Title: A new LNC89/LNC60-Col11a2 axis revealed by whole-transcriptome analysis may be associated with goiters related to excess iodine nutrition

doi: 10.3389/fendo.2024.1407859

Figure Lengend Snippet: The expression of LNC60 and Col11a2 mRNA in the peripheral blood of Goiter patients and health control populations. *** P< 0.001, **** P< 0.0001, t test.

Article Snippet: Primary antibodies are rabbit anti-mouse Col11a2 antibody (YT1009, 1:500, Immunoway, China) and mouse anti-mouse β-actin antibody (TA811000, 1:2000, OriGene, USA) while mouse and rabbit second antibodies from Abbkine (A25012, 1:10000, China) and CST (14708, 1:2000, USA).

Techniques: Expressing, 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) 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: Molecular and Cellular Biology

Article Title: A Multifunctional Domain in Human CRM1 (Exportin 1) Mediates RanBP3 Binding and Multimerization of Human T-Cell Leukemia Virus Type 1 Rex Protein

doi: 10.1128/mcb.23.23.8751-8761.2003

Figure Lengend Snippet: FIG. 1. The amino acid residues of hCRM1 required for Rex ac- tivity. (A) The ability of CRM1s to support Rex activity. HeLa cells were transfected with the indicated plasmids. After cell lysis, the amount of CAT and the activity of -Gal were measured and CAT/ -Gal ratios were calculated. The ratio for the control sample without pSRTAgRexM64 and pSRCRM1s was arbitrarily set at 1. The amount of CAT and the -Gal activity in control samples were over 300 pg and 3.0 103 U, respectively. Error bars represent standard deviations. (B) Restoration of Rex activity by overexpression of CRM1s in HeLa cells. The experimental procedure was the same as that described for panel A. (C) Effect of overexpressing CRM1s on Rex activity in REF52 cells. REF52 cells were transfected with the indicated plasmids. At 24 h of posttransfection, CAT/-Gal ratios were calculated. The ratio for the sample without pSRCRM1s was arbi- trarily set at 1. (D) Western blot analysis of various CRM1s. A fraction of each sample used in the experiments described for panel C was subjected to Western blot analysis using the anti-hCRM1 antibody to examine CRM1 protein synthesis. This antibody was raised with the peptide, which represents the carboxy-terminal region of hCRM1 and has a sequence different from that of rCRM1, so it does not recognize endogenous rCRM1. (E) Effect of CRM1s on Rex-mediated Gag expression from HTLV-1 molecular clone. At 48 h posttransfection, Gag/-Gal ratios were calculated. The ratio for the sample without pSRCRM1s was arbitrarily set at 1. (F) A schematic representation of hCRM1 functional domains. The amino acid sequence of hCRM1 is shown. Experiments characterizing the RanGTP binding domain (bro- ken line) (33), LMB binding residue (arrowhead) (24), Rev-interacting amino acids (asterisks) (2), and the domain binding to NES (under- line) (33) have been previously reported. The residues 411, 414, 474, and 481 in hCRM1 are indicated in bold characters, and the corre- sponding residues of rCRM1 are indicated under the hCRM1 se- quence in single-letter amino acid code.

Article Snippet: Mouse anti-GAL4 monoclonal antibody (Santa Cruz Biotechnology) and affinity-purified chicken antihCRM1 antibody (18) were used as primary antibodies to detect GAL-fused proteins and CRM1s, respectively.

Techniques: Activity Assay, Transfection, Lysis, Control, Over Expression, Western Blot, Sequencing, Expressing, Functional Assay, Binding Assay, Residue

Journal: Molecular and Cellular Biology

Article Title: A Multifunctional Domain in Human CRM1 (Exportin 1) Mediates RanBP3 Binding and Multimerization of Human T-Cell Leukemia Virus Type 1 Rex Protein

doi: 10.1128/mcb.23.23.8751-8761.2003

Figure Lengend Snippet: FIG. 2. In vivo interaction of Rex with CRM1 mutants in which one amino acid is replaced. REF52 cells were transfected with the plasmid expressing GAL-CRM1s in combination with pRexVP, pG5BCAT, and pCDM-gal. The cells were harvested and subjected to CAT and -Gal assays, and CAT/-Gal ratios were calculated. The ratio for the control sample, which detected the interaction between GAL-hCRM1 and Rex-VP, was arbitrarily set at 1. The amount of CAT and -Gal activity in control samples were over 400 pg and 3.0 103 U, respec- tively. GAL4 nonfusion protein, expressing only a GAL4 region, was used as a negative control. A fraction of each sample was subjected to Western blot analysis using the anti-GAL4 monoclonal antibody to examine GAL-CRM1 expression.

Article Snippet: Mouse anti-GAL4 monoclonal antibody (Santa Cruz Biotechnology) and affinity-purified chicken antihCRM1 antibody (18) were used as primary antibodies to detect GAL-fused proteins and CRM1s, respectively.

Techniques: In Vivo, Transfection, Plasmid Preparation, Expressing, Control, Activity Assay, Negative Control, Western Blot

Journal: Molecular and Cellular Biology

Article Title: A Multifunctional Domain in Human CRM1 (Exportin 1) Mediates RanBP3 Binding and Multimerization of Human T-Cell Leukemia Virus Type 1 Rex Protein

doi: 10.1128/mcb.23.23.8751-8761.2003

Figure Lengend Snippet: FIG. 6. Binding characterization of two amino acid-substituted CRM1s. (A) In vivo interaction of CRM1s with Rex. REF52 cells were treated as described for Fig. 2 except for the plasmids that expressed two amino acid-substituted CRM1s as a GAL4 fusion protein. A portion of each sample was subjected to Western blot analysis to confirm GAL-CRM1 expression. (B) In vitro binding of h411/414 to RanBP3. As shown in Fig. 5A, in vitro-translated CRM1s were incubated with GST or GST-RanBP3 immobilized on glutathione-Sepharose 4B.

Article Snippet: Mouse anti-GAL4 monoclonal antibody (Santa Cruz Biotechnology) and affinity-purified chicken antihCRM1 antibody (18) were used as primary antibodies to detect GAL-fused proteins and CRM1s, respectively.

Techniques: Binding Assay, In Vivo, Western Blot, Expressing, In Vitro, Incubation

Journal: Histochemistry and Cell Biology

Article Title: Multispectral imaging reveals the tissue distribution of tetraspanins in human lymphoid organs

doi: 10.1007/s00418-015-1326-2

Figure Lengend Snippet: Expression of CD37 and CD53 on immune cell subsets in blood. a Flow cytometry analysis of expression of CD37 or CD53 ( black line ) on CD4 and CD8 T cells, B cells, monocytes and NK cells versus isotype control ( gray line ). Gating strategy is presented in Supplementary Figure 1. Expression levels of CD37 ( b ) and CD53 ( c ) were normalized for isotype staining by background subtraction. Experiments were performed with PBLs from three healthy donors. Data present mean ± SD. d Flow cytometry analysis of expression of CD37 or CD53 ( black line ) on mDCs (BDCA1 + CD19 − ) or pDCs (BDCA2 + ) versus isotype control ( gray line ). Expression levels of CD37 ( e ) and CD53 ( f ) were normalized for isotype staining by background subtraction. Experiments were performed with PBLs from two healthy donors. Data present mean ± SD

Article Snippet: Single-cell suspensions were first stained with primary antibodies against human CD53 (mem53, Serotec), CD37 (WR17, home-made) or isotype controls in PBS with 1 % BSA and 0.05 % NaN 3 (PBA) supplemented with 2 % HS for 30 min at 4 °C, followed by incubation with goat-anti-mouse Alexa488 antibody (Molecular Probes).

Techniques: Expressing, Flow Cytometry, Control, Staining

Journal: Histochemistry and Cell Biology

Article Title: Multispectral imaging reveals the tissue distribution of tetraspanins in human lymphoid organs

doi: 10.1007/s00418-015-1326-2

Figure Lengend Snippet: Subcellular localization of CD37 and CD53. Localization of a CD37 or b CD53 ( green ) in monocytes was studied by dual staining with calreticulin (ER), syntaxin 13 (endosomes) or Lamp1 (lysosomes) ( red ). Merge: co-localization in yellow ( white arrows ). Scale bar 5 μm

Article Snippet: Single-cell suspensions were first stained with primary antibodies against human CD53 (mem53, Serotec), CD37 (WR17, home-made) or isotype controls in PBS with 1 % BSA and 0.05 % NaN 3 (PBA) supplemented with 2 % HS for 30 min at 4 °C, followed by incubation with goat-anti-mouse Alexa488 antibody (Molecular Probes).

Techniques: Staining

Journal: Histochemistry and Cell Biology

Article Title: Multispectral imaging reveals the tissue distribution of tetraspanins in human lymphoid organs

doi: 10.1007/s00418-015-1326-2

Figure Lengend Snippet: Expression of CD37 and CD53 on human spleen. a Original multispectral image of human spleen stained for CD37 (Alexa488), CD53 (Alexa568) and cell nuclei (DAPI). Scale bar 100 μm. Composite RGB image ( b ) of unmixed CD37 ( c in green ), CD53 ( d in red ) and DAPI ( e in blue ) signal after correction for autofluorescence. One representative image is shown

Article Snippet: Single-cell suspensions were first stained with primary antibodies against human CD53 (mem53, Serotec), CD37 (WR17, home-made) or isotype controls in PBS with 1 % BSA and 0.05 % NaN 3 (PBA) supplemented with 2 % HS for 30 min at 4 °C, followed by incubation with goat-anti-mouse Alexa488 antibody (Molecular Probes).

Techniques: Expressing, Staining

Journal: Histochemistry and Cell Biology

Article Title: Multispectral imaging reveals the tissue distribution of tetraspanins in human lymphoid organs

doi: 10.1007/s00418-015-1326-2

Figure Lengend Snippet: Spectral imaging analysis of human spleen stained for CD20 (Warp Red), CD53 (True Blue) and cell nuclei (Nuclear Red). a A spectral library of three chromogens (Warp Red ( red line ), True Blue ( blue line ) and Nuclear Red ( green line )) was built in Nuance software using single-stained human spleen tissues. b Representative original multispectral image. Scale bar 100 μm. Composite RGB image ( c ) of unmixed CD20 ( d in red ), CD53 ( e in blue ) and nuclei ( f in green in composite RGB image) signal. g Tissue segmentation; B cell follicle (B, yellow ), red pulp (RP, green ) and other tissue (blood vessels, collagen; blue ). h Segmentation of individual cells ( green ) based on Nuclear Red staining. i Thresholds for Warp Red and True Blue staining were set to score CD20 − CD53 dim ( blue ), CD20 + CD53 dim ( red ), CD20 + CD53 bright ( yellow ) or CD20 − CD53 bright ( green ) cells. j–l Scatter plots showing optical densities for CD20 (Y-axis) and CD53 (X-axis) of individual cells in B cell follicles ( j , l blue ) and red pulp ( k–l red ) and thresholds used for scoring ( dotted lines ). A representative of 2000 cells per tissue region is plotted

Article Snippet: Single-cell suspensions were first stained with primary antibodies against human CD53 (mem53, Serotec), CD37 (WR17, home-made) or isotype controls in PBS with 1 % BSA and 0.05 % NaN 3 (PBA) supplemented with 2 % HS for 30 min at 4 °C, followed by incubation with goat-anti-mouse Alexa488 antibody (Molecular Probes).

Techniques: Imaging, Staining, Software

Journal: Histochemistry and Cell Biology

Article Title: Multispectral imaging reveals the tissue distribution of tetraspanins in human lymphoid organs

doi: 10.1007/s00418-015-1326-2

Figure Lengend Snippet: Localization and expression of CD53 in bone marrow ( a–f ), spleen ( g–l ) and appendix ( m–r ). a , g , m Representative original multispectral image of lymphoid organ stained for CD20 (Warp Red), CD53 (True Blue) and cell nuclei (Nuclear Red). Scale bars in a , g , m = 100 μm. b , h , n , Composite RGB image after spectral unmixing of original image ( red CD20, blue CD53, green nuclei). c , i , o , Image showing scoring of CD20 − CD53 dim ( blue ), CD20 + CD53 dim ( red ), CD20 + CD53 bright ( yellow ) or CD20 − CD53 bright ( green ) cells. d , j , p , Optical density of CD53 on individual cells in human bone marrow ( d ), in B cell follicle ( blue line ) and in T cell zone and in red pulp ( red line ) in human spleen ( j ) and in B cell follicle ( blue line ) and in lamina propria ( red line ) in human appendix ( p ). Optical densities were binned per 0.05 and normalized to % of max. Percentage of CD53 dim and CD53 bright cells in the CD20 + cell population ( e ) and in the CD3 + cell population ( f ) in human bone marrow. Percentage of CD53 bright cells in the CD20 + cell population in the B cell follicle and red pulp ( k ), and in the CD3 + cell population in the T cell zone and red pulp ( l ) in human spleen. Percentage of CD53 bright cells in the CD20+ cell population ( q ) and in the CD3 + cell population ( r ) in the B cell follicle and lamina propria in human appendix. Each dot represents data of one ×20 image from the lymphoid tissue. The red line represents the mean. * P < 0.05, **** P < 0.0001

Article Snippet: Single-cell suspensions were first stained with primary antibodies against human CD53 (mem53, Serotec), CD37 (WR17, home-made) or isotype controls in PBS with 1 % BSA and 0.05 % NaN 3 (PBA) supplemented with 2 % HS for 30 min at 4 °C, followed by incubation with goat-anti-mouse Alexa488 antibody (Molecular Probes).

Techniques: Expressing, Staining

Journal: Virology

Article Title: Human cytomegalovirus-infected cells release extracellular vesicles that carry viral surface proteins

doi: 10.1016/j.virol.2018.08.008

Figure Lengend Snippet: (A) stain-free blot representing the spectrum of proteins obtained for HCMV and EV fractions after separation of AD169 HCMV pre-paration using a iodixanol step-gradient centrifugation. (B) Detection of EV-associated proteins CD63 (~60 kDa), Rab27A (~30 kDa), and calnexin (~25 kDa) proteins in both fractions. (C) Detection of HCMV capsid protein MCP (~150 kDa) and HCMV tegument protein, pp150 (~160 kDa) in HCMV and EV fractions after separation of AD169 viral preparation using a iodixanol step-gradient centrifugation.

Article Snippet: 10 μg of proteins were loaded on a 4–20% precast polyacrylamide gel (Bio-Rad Laboratories, Hercules, CA) and separated by SDS-PAGE, then transferred to PVDF membranes and probed with anti-CD63 (1 μg/ml, Thermo Fisher Scientific, Waltham, MA), anti-Calnexin (1 μg/ml, Thermo Fisher Scientific, Waltham, MA), anti-Rab27A (1 μg/ml, Thermo Fisher Scientific, Waltham, MA), anti-HCMV capsid MCP (2 μg/ml), and anti-HCMV tegument pp150 (2 μg/ml) monoclonal primary anti-mouse monoclonal antibodies (clones 28–4 and 36–14) and then goat peroxidase-conjugated anti-mouse IgG secondary anti-body (Bio-Rad Laboratories, Hercules, CA).

Techniques: Staining, Gradient Centrifugation