Journal: Bioengineered

Article Title: Long non-coding RNA TUG1 knockdown prevents neurons from death to alleviate acute spinal cord injury via the microRNA-338/BIK axis

doi: 10.1080/21655979.2021.1966258

Figure Lengend Snippet: List of siRNA and inhibitor sequences

Article Snippet: The mixture of protein extracts from PC12 and AGE1.HN cells and 50 pmol biotinylated TUG1 was incubated for 1 h with 50 μL streptavidin agarose beads (Life Technologies) at 4°C or with biotinylated arbitrary single nucleotide as negative control (NC).

Techniques: Sequencing

Journal: Bioengineered

Article Title: Long non-coding RNA TUG1 knockdown prevents neurons from death to alleviate acute spinal cord injury via the microRNA-338/BIK axis

doi: 10.1080/21655979.2021.1966258

Figure Lengend Snippet: Primer sequence used in this study

Article Snippet: The mixture of protein extracts from PC12 and AGE1.HN cells and 50 pmol biotinylated TUG1 was incubated for 1 h with 50 μL streptavidin agarose beads (Life Technologies) at 4°C or with biotinylated arbitrary single nucleotide as negative control (NC).

Techniques: Sequencing

Journal: Bioengineered

Article Title: Long non-coding RNA TUG1 knockdown prevents neurons from death to alleviate acute spinal cord injury via the microRNA-338/BIK axis

doi: 10.1080/21655979.2021.1966258

Figure Lengend Snippet: TUG1 expresses highly in SCI tissues and AGE1.HN and PC12 cells. Rats were categorized into sham group (n = 5) and ASCI group (n = 5). (a) BBB scores were evaluated (unpaired t test, ***p < 0.001; t(8) = 17.574, p < 0.001). (b) TUG1 expression in spinal cord tissues of ASCI and sham-operated rats determined by RT-qPCR normalized to GAPDH (unpaired t test, ***p < 0.001; t(5.414) = −19.860, p < 0.001). (c) the protein expression of HIF-1α in AGE1.HN and PC12 cells treated with and without hypoxia measured by western blot analysis normalized to β-actin (unpaired t test, ***p < 0.001, AGE1.HN: t(4.354) = −34.359, p < 0.001; PC12: t(4.453) = −22.790, p < 0.001). (d) TUG1 expression in AGE1.HN and PC12 cells treated with and without hypoxia measured by RT-qPCR normalized to GAPDH (unpaired t test, ***p < 0.001. AGE1.HN: t(4.215) = −44.328, p < 0.001; PC12: t(4.879) = −37.856, p < 0.001). Data were displayed as the mean ± SD. Five independent assays were conducted only for in vitro experiments

Article Snippet: The mixture of protein extracts from PC12 and AGE1.HN cells and 50 pmol biotinylated TUG1 was incubated for 1 h with 50 μL streptavidin agarose beads (Life Technologies) at 4°C or with biotinylated arbitrary single nucleotide as negative control (NC).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, In Vitro

Journal: Bioengineered

Article Title: Long non-coding RNA TUG1 knockdown prevents neurons from death to alleviate acute spinal cord injury via the microRNA-338/BIK axis

doi: 10.1080/21655979.2021.1966258

Figure Lengend Snippet: TUG1 promotes AGE1.HN and PC12 cell death. (a) the protein expression of c-caspase3 and c-caspase3/total caspase3 in AGE1.HN and PC12 cells treated with and without hypoxia measured by western blot analysis normalized to β-actin (unpaired t test, ***p < 0.001. For c-caspase3: AGE1.HN: t(5.061) = −35.105, p < 0.001; PC12: t(8) = −25.322, p < 0.001. For c-caspase3/total caspase3: AGE1.HN: t(8) = −49.07, p < 0.001; PC12: t(8) = −18.78, p < 0.001). Human or rat pcDNA-TUG1 and siRNA-TUG1 and their NCs were transfected into hypoxia-treated AGE1.HN and PC12 cells, respectively. (b) TUG1 expression in AGE1.HN and PC12 cells after transfection measured by RT-qPCR normalized to GAPDH (one-way ANOVA, ***p < 0.001. AGE1.HN: F(3,16) = 35,058.432, p < 0.001; PC12: F(3,16) = 70,046.402, p < 0.001). (c) the protein expression of c-caspase3 and c-caspase3/total caspase3 in AGE1.HN and PC12 cells after transfection measured by western blot analysis normalized to β-actin (one-way ANOVA, ***p < 0.001. For c-caspase3: AGE1.HN: F(3,16) = 416.825, p < 0.001; PC12: F(3,16) = 260.449, p < 0.001. For c-caspase3/total caspase3: AGE1.HN: F(3,16) = 475.2, p < 0.001; PC12: F(3,16) = 468.6, p < 0.001). (d) the death rate of AGE1.HN and PC12 cells after transfection measured by flow cytometry (one-way ANOVA, *p = 0.019, **p = 0.005, ***p < 0.001. AGE1.HN: F(3,16) = 184.670, p < 0.001; PC12: F(3,16) = 34.701, p < 0.001). (e) the distribution of TUG1 in AGE1.HN and PC12 cells evaluated by nuclear/cytoplasmic fractionation. Data were displayed as the mean ± SD. Five independent assays were conducted only for in vitro experiments

Article Snippet: The mixture of protein extracts from PC12 and AGE1.HN cells and 50 pmol biotinylated TUG1 was incubated for 1 h with 50 μL streptavidin agarose beads (Life Technologies) at 4°C or with biotinylated arbitrary single nucleotide as negative control (NC).

Techniques: Expressing, Western Blot, Transfection, Quantitative RT-PCR, Flow Cytometry, Fractionation, In Vitro

Journal: Bioengineered

Article Title: Long non-coding RNA TUG1 knockdown prevents neurons from death to alleviate acute spinal cord injury via the microRNA-338/BIK axis

doi: 10.1080/21655979.2021.1966258

Figure Lengend Snippet: TUG1 downregulates miR-338 expression in AGE1.HN and PC-12 cells. (a) screening of differentially expressed miRNAs in spinal cord tissue of SCI rats by GEO database. (b) the intersection of potential binding miRNAs for TUG1 and downregulated miRNAs in the GEO database. (c) expression of miR-338 in spinal cord tissues of SCI rats as queried in GEO database. (d) the potential binding relationship between TUG1 and miR-338 predicted by RNA22. (e) miR-338 expression in rat tissues (n = 5) and in AGE1.HN and PC12 cells treated with hypoxia (unpaired t test, ***p < 0.001; rat: t(8) = 15.067, p < 0.001: AGE1.HN: t(4.693) = 27.339, p < 0.001; PC12: t(8) = 29.803, p < 0.001). (f) AGO2 antibody precipitate was enriched in TUG1 and miR-338 verified by RIP normalized to IgG (one-way ANOVA, ***p < 0.001. AGE1.HN: F(2,12) = 2698.390, p < 0.001, F(2,12) = 5214.375, p < 0.001; PC12: F(2,12) = 3525.508, p < 0.001, F(2,12) = 1959.624, p < 0.001). (g) the enrichment of miR-338 in PC-12 cells by TUG1 determined by RNA pull-down normalized to IgG (one-way ANOVA, ***p < 0.001. AGE1.HN: F(3,16) = 2787.589, p < 0.001; PC12: F(3,16) = 956.353, p < 0.001). Data were displayed as the mean ± SD. Five independent assays were conducted only for in vitro experiments

Article Snippet: The mixture of protein extracts from PC12 and AGE1.HN cells and 50 pmol biotinylated TUG1 was incubated for 1 h with 50 μL streptavidin agarose beads (Life Technologies) at 4°C or with biotinylated arbitrary single nucleotide as negative control (NC).

Techniques: Expressing, Binding Assay, In Vitro

Journal: Bioengineered

Article Title: Long non-coding RNA TUG1 knockdown prevents neurons from death to alleviate acute spinal cord injury via the microRNA-338/BIK axis

doi: 10.1080/21655979.2021.1966258

Figure Lengend Snippet: TUG1 inhibits miR-338 expression to promote AGE1.HN and PC12 cell death. (a) miR-338 expression in AGE1.HN and PC-12 cells in response to pcDNA-TUG1 or siRNA-TUG1 determined by RT-qPCR normalized to GAPDH (one-way ANOVA, ***p < 0.001. AGE1.HN: F(3,16) = 1143.706, p < 0.001; PC12: F(3,16) = 1053.589, p < 0.001). (b) miR-338 expression in AGE1.HN and PC-12 cells in response to pcDNA-TUG1 or pcDNA-TUG1 + miR-338 mimic determined by RT-qPCR normalized to GAPDH (one-way ANOVA, ***p < 0.001. AGE1.HN: F(3,16) = 1168.594, p < 0.001; PC12: F(3,16) = 1667.774, p < 0.001). (c) cell death in AGE1.HN and PC-12 cells in response to pcDNA-TUG1 or pcDNA-TUG1 + miR-338 mimic determined by flow cytometry (one-way ANOVA, **P = 0.002, ***p < 0.001. AGE1.HN: F(3,16) = 117.167, p < 0.001; PC12: F(3,16) = 173.777, p < 0.001). Data were displayed as the mean ± SD. Five independent assays were conducted only for in vitro experiments

Article Snippet: The mixture of protein extracts from PC12 and AGE1.HN cells and 50 pmol biotinylated TUG1 was incubated for 1 h with 50 μL streptavidin agarose beads (Life Technologies) at 4°C or with biotinylated arbitrary single nucleotide as negative control (NC).

Techniques: Expressing, Quantitative RT-PCR, Flow Cytometry, In Vitro

Journal: Bioengineered

Article Title: Long non-coding RNA TUG1 knockdown prevents neurons from death to alleviate acute spinal cord injury via the microRNA-338/BIK axis

doi: 10.1080/21655979.2021.1966258

Figure Lengend Snippet: The regulatory role of TUG1/miR-338/BIK axis on rats with SCI (n = 5). (a) the BBB scores for hindlimb locomotion in the rats with ASCI injected with siRNA-TUG1 alone or with miR-338 inhibitor (one-way ANOVA, **p = 0.008, ***p < 0.001. F(3,16) = 175.553, p < 0.001). (b) detection of cell death in rat spinal cord by TUNEL staining (one-way ANOVA, ***p < 0.001. F(5,24) = 296.154, p < 0.001). (c) TUG1 (normalized to GAPDH) and miR-338 expression (normalized to U6) as well as the BIK mRNA expression (normalized to GAPDH) in spinal cord tissues of rats determined by RT-qPCR (one-way ANOVA, ***p < 0.001. TUG1: F(3,16) = 140.714, p < 0.001; miR-338: F(3,16) = 291.155, p < 0.001; BIK: F(3,16) = 251.190, p < 0.001). (d) the protein expression of c-caspase3 and c-caspase3/total caspase3 in spinal cord tissues of rats measured by western blot analysis normalized to β-actin (one-way ANOVA, ***p < 0.001. For c-caspase3/total caspase3: F(3,16) = 146.102, p < 0.001. For c-caspase3/total caspase3: F(3,16) = 702.9, p < 0.001). Data were displayed as the mean ± SD. Five independent assays were conducted only for in vitro experiments

Article Snippet: The mixture of protein extracts from PC12 and AGE1.HN cells and 50 pmol biotinylated TUG1 was incubated for 1 h with 50 μL streptavidin agarose beads (Life Technologies) at 4°C or with biotinylated arbitrary single nucleotide as negative control (NC).

Techniques: Injection, TUNEL Assay, Staining, Expressing, Quantitative RT-PCR, Western Blot, In Vitro

Journal: Bioengineered

Article Title: Long non-coding RNA TUG1 knockdown prevents neurons from death to alleviate acute spinal cord injury via the microRNA-338/BIK axis

doi: 10.1080/21655979.2021.1966258

Figure Lengend Snippet: Mechanisms of TUG1 mediated SCI progression. TUG1 interacts with miR-338, leading to BIK activation, which accelerates SCI via promoting cell death. While TUG1 knockdown downregulates BIK expression by interacting with miR-338, thus lowering c-caspase3 to hamper death

Article Snippet: The mixture of protein extracts from PC12 and AGE1.HN cells and 50 pmol biotinylated TUG1 was incubated for 1 h with 50 μL streptavidin agarose beads (Life Technologies) at 4°C or with biotinylated arbitrary single nucleotide as negative control (NC).

Techniques: Activation Assay, Knockdown, Expressing

Journal: Cancer Immunology, Immunotherapy

Article Title: Blockade of programmed death-1/programmed death ligand pathway enhances the antitumor immunity of human invariant natural killer T cells

doi: 10.1007/s00262-016-1901-y

Figure Lengend Snippet: Proliferation of human iNKT cells with PDL1 blockade. PBMCs were obtained from six healthy donors and eight non-small cell lung cancer patients. On day 0, PBMCs were stimulated with αGalCer-pulsed IL-2/GM-CSF cultured APCs with anti-PDL1 antibody or isotype control. On day 7, cells were collected and restimulated with PDL1-blocked or isotype control-treated APCs at a ratio of 1:2.5. The cells were collected and counted on day 14, and the proportion of Vα24 + Vβ11 + iNKT cells was analyzed using flow cytometry. a Anti-PDL1 antibody binding and PDL1 positivity on APCs were assessed using anti-mouse biotin plus streptavidin staining. b The percentage of PDL1-positive iNKT cells on days 0 and 7 were analyzed with APC-conjugated anti-human PDL1. The gray-shaded histogram represents the isotype control; the unshaded histogram represents PDL1. c The number of Vα24 + Vβ11 + iNKT cells on day 7 is shown. PDL1 positivity on APCs was analyzed according to the population comparison method using the FlowJo software program. P values were calculated using the unpaired t test. isotype, isotype control; aPDL1 ab, anti-PDL1 antibody

Article Snippet: Peroxidase streptavidin (Jackson ImmunoResearch) was used in an ELISA to determine the concentration of anti-PDL1 antibodies.

Techniques: Cell Culture, Control, Flow Cytometry, Binding Assay, Staining, Comparison, Software

Journal: Scientific Reports

Article Title: Effect of human very low-density lipoproteins on cardiotrophin-like cytokine factor 1 (CLCF1) activity

doi: 10.1038/s41598-018-22400-y

Figure Lengend Snippet: CLCF1 binds recombinant and serum ApoE. ( A ) Recombinant CLCF1 (200 ng) and ApoE2, 3 or 4 (250 ng) were incubated alone or in combination for 16 h. The samples were subjected to immunoprecipitation (IP) with anti-CLCF1 and protein G agarose. Immunoprecipitated proteins were analyzed by WB using mAbs specific for ApoE (upper panel) or CLCF1 (lower panel). Lanes “CLC 2 ng” and “ApoE3 20 ng”, show results of recombinant proteins directly subjected to SDS-PAGE and WB blot analysis. ( B ) CLCF1 and ApoE form a complex when co-expressed in HEK-293 cells. The cell culture medium from stable transfectants expressing the indicated proteins in combination with CRLF1 or from cells transfected with empty expression vector (lane D5) was subjected to immunoaffinity chromatography with rat-anti-human ApoE and anti-rat Ig Agarose. The eluates were analysed by WB using anti-ApoE or anti-CLC mAbs (upper panels). Supernatants of the stable transfectants were analyzed for the presence of ApoE or CLCF1 by WB (lower panels). ( C and D ) CLCF1 binds mouse and human serum ApoE. ( C ) Mouse serum samples isolated from WT or ApoE −/− mice were diluted 1:20, mixed with biotinylated mouse CLCF1 and the complexes immunoprecipitated with rabbit anti-mouse ApoE and anti-rabbit Ig agarose. The eluates were analysed for ApoE or CLCF1 by WB using anti-mouse ApoE or HRP-labelled streptavidin. The last two lanes show the signals obtained with 40 ng of biotinylated mouse CLCF1 or ApoE included as control. ( D ) Human serum samples were diluted 1:20, mixed with biotinylated human CLCF1 and the complexes immunoprecipitated with rat anti-ApoE and anti-rat Ig agarose. The eluates were analyzed for ApoE or CLCF1 by WB using anti-ApoE or HRP-labelled streptavidin. The last two lanes show the signals obtained with 40 ng of biotinylated human CLCF1 or ApoE included as control. Blots’ images where cropped to show relevant areas.

Article Snippet: Eluates were analyzed by WB using HRP-streptavidin (GE-Healthcare Life Science, Cedarlane, Burlington, ON) or rabbit anti-mouse ApoE Ab (Abcam Inc, Cedarlane) followed by HRP-labelled TrueBlot™ anti-rabbit Ab (Rockland Immunochemicals Inc).

Techniques: Recombinant, Incubation, Immunoprecipitation, SDS Page, Cell Culture, Expressing, Transfection, Plasmid Preparation, Chromatography, Isolation

Journal: Scientific Reports

Article Title: Effect of human very low-density lipoproteins on cardiotrophin-like cytokine factor 1 (CLCF1) activity

doi: 10.1038/s41598-018-22400-y

Figure Lengend Snippet: AlphaLISA proximity assay shows CLCF1-ApoE complex formation that is increased by the lipid DMPC. ( A ) ApoE2, 3 or 4 were mixed with CLCF1 (10 nM) in PBS 0.1% BSA for 1 h with IgG rat anti-ApoE (3 nM) and anti-IgG rat acceptor beads (10 μg/mL). Streptavidin donor beads (40 μg/mL) were further added for 1 h and 615 nm fluorescence signal assessed. ( B ) The indicated ApoE isotypes (30 nM) were mixed at 37 °C with increasing amount of DMPC for 1 h. ApoE-DMPC mix were further incubated with CLCF1 and subjected to AlpahLISA proximity assay. Histograms indicate the mean fluorescence intensity of 3 independent samples ± SD.

Article Snippet: Eluates were analyzed by WB using HRP-streptavidin (GE-Healthcare Life Science, Cedarlane, Burlington, ON) or rabbit anti-mouse ApoE Ab (Abcam Inc, Cedarlane) followed by HRP-labelled TrueBlot™ anti-rabbit Ab (Rockland Immunochemicals Inc).

Techniques: Proximity Assay, Fluorescence, Incubation

Journal: Scientific Reports

Article Title: Effect of human very low-density lipoproteins on cardiotrophin-like cytokine factor 1 (CLCF1) activity

doi: 10.1038/s41598-018-22400-y

Figure Lengend Snippet: ApoE3 increases CLCF1-induced CNTFR activation. ( A ) Ba/F3 transfected with CNTFRα, gp130 and LIFRβ cDNA were successively incubated with mouse biotinylated CLCF1 (1 μg/ml) alone or in the presence of ApoE3 (10 mg/ml) and PE-labelled streptavidin and fluorescence was assessed by flow cytometry. Vehicle: cells incubated with PE-streptavidin alone. ( B ) Ba/F3-CNTFR were stimulated with CLCF1 (100 ng/ml) or ApoE3 (10 mg/ml) alone or in combination for 15 min. STAT3 tyrosine phosphorylation was assessed by intracellular staining with anti-pSTAT3 mAb and flow cytometry. Vehicle: cells incubated with PE-streptavidin alone. Histograms indicate the mean fluorescence intensity of 3 independent samples ± SD.

Article Snippet: Eluates were analyzed by WB using HRP-streptavidin (GE-Healthcare Life Science, Cedarlane, Burlington, ON) or rabbit anti-mouse ApoE Ab (Abcam Inc, Cedarlane) followed by HRP-labelled TrueBlot™ anti-rabbit Ab (Rockland Immunochemicals Inc).

Techniques: Activation Assay, Transfection, Incubation, Fluorescence, Flow Cytometry, Staining

Journal: Scientific Reports

Article Title: Effect of human very low-density lipoproteins on cardiotrophin-like cytokine factor 1 (CLCF1) activity

doi: 10.1038/s41598-018-22400-y

Figure Lengend Snippet: CLCF1 co-purifies or co-elutes with serum lipoprotein complexes. ( A ) WT mouse, ( B ) human or ( C ) ApoE −/− mouse sera were mixed with corresponding biotinylated CLCF1. The mixtures were fractionated by FPLC using a Superose 6 HR 10/ column in order to obtain 40 fractions. The fractions were analyzed for ApoE or CLCF1 by WB using anti-ApoE or HRP-labelled streptavidin. The right lanes show the signals obtained with 40 ng of ApoE or biotinylated CLCF1 included as control. Blots’ images where cropped to show relevant areas.

Article Snippet: Eluates were analyzed by WB using HRP-streptavidin (GE-Healthcare Life Science, Cedarlane, Burlington, ON) or rabbit anti-mouse ApoE Ab (Abcam Inc, Cedarlane) followed by HRP-labelled TrueBlot™ anti-rabbit Ab (Rockland Immunochemicals Inc).

Techniques:

Journal: Scientific Reports

Article Title: Effect of human very low-density lipoproteins on cardiotrophin-like cytokine factor 1 (CLCF1) activity

doi: 10.1038/s41598-018-22400-y

Figure Lengend Snippet: Biotinylated LDL and VLDL bind CLCF1. ( A ) Interaction between CLCF1 and lipoproteins was assessed using ligand blotting assays: 0.1–0.2 μg of hCLC, or 1 μg of BSA were subjected to non-reducing SDS-PAGE. Proteins were revealed by Coomassie blue staining (left section) or electro-transferred to nitrocellulose and incubated successively with 30 μg/ml biotinylated VLDL, LDL or HDL and HRP-labeled neutravidin (middle 3 panels). Signal was revealed by chemiluminescence. Image in the far right section shows results of incubation with HRP-labeled neutravidin alone. Lane hCLC biot; 40 ng of biotinylated CLC was used as positive control for neutravidin detection. Blots’ images where cropped to show relevant areas. ( B ) The formation of LDL and VLDL complexes can be detected using an AlphaLISA proximity assay. Biotinylated hCLCF1 was mixed with LDL, HDL and VLDL covalently coupled to acceptor beads (10 μg/mL). Streptavidin donor beads (40 μg/mL) were further added for 1 h and 615 nm fluorescence signal assessed. Histograms indicate the mean fluorescence intensity of 3 independent samples ± SD.

Article Snippet: Eluates were analyzed by WB using HRP-streptavidin (GE-Healthcare Life Science, Cedarlane, Burlington, ON) or rabbit anti-mouse ApoE Ab (Abcam Inc, Cedarlane) followed by HRP-labelled TrueBlot™ anti-rabbit Ab (Rockland Immunochemicals Inc).

Techniques: SDS Page, Staining, Incubation, Labeling, Positive Control, Proximity Assay, Fluorescence

Journal: Scientific Reports

Article Title: Effect of human very low-density lipoproteins on cardiotrophin-like cytokine factor 1 (CLCF1) activity

doi: 10.1038/s41598-018-22400-y

Figure Lengend Snippet: VLDL increases CLCF1 binding to CNTFR-expressing cells. Ba/F3 transfected with CNTFRα, gp130 and LIFRβ cDNA, IMR32 neuroblastoma or 3T3-L1 pre-adipocytes were successively incubated with species-matched biotinylated CLCF1 (1 μg/ml) alone or in the presence of LDL, HDL or VLDL (all at 100 μg/ml). Cells were then incubated with PE-conjugated streptavidin and fluorescence was assessed by flow cytometry. Vehicle: cells incubated with PE-streptavidin alone. Histograms indicate the mean fluorescence intensity of 3 independent samples ± SD.

Article Snippet: Eluates were analyzed by WB using HRP-streptavidin (GE-Healthcare Life Science, Cedarlane, Burlington, ON) or rabbit anti-mouse ApoE Ab (Abcam Inc, Cedarlane) followed by HRP-labelled TrueBlot™ anti-rabbit Ab (Rockland Immunochemicals Inc).

Techniques: Binding Assay, Expressing, Transfection, Incubation, Fluorescence, Flow Cytometry

Journal: mAbs

Article Title: Mimicking the germinal center reaction in hybridoma cells to isolate temperature-selective anti-PEG antibodies

doi: 10.4161/mabs.29124

Figure Lengend Snippet: Figure 2. Analysis of membrane immunoglobulin on hybridoma cells. ( A ) Membrane-bound immunoglobulin on hybridoma and FO myeloma cells was detected by flow cytometry after staining the cells with goat anti-mouse Ig and rabbit anti-goat FITC. ( B ) Functional antigen-binding of membrane immunoglobulin on 3.3 anti-PEG and anti-β-glucuronidase 7G8 hybridoma cells was detected on a flow cytometer with biotinylated PEG or β-glucuronidase antigens followed by Alexa Fluor 647-conjugated streptavidin. ( C ) 7G8 anti-β-glucuronidase and 3.3 anti-PEG hybridoma cells mixed at a ratio of 99:1 were stained with 0.5 nM biotin-PEG and Alexa Fluor 647-conjugated streptavidin. Hybridoma cells that bound biotin-PEG (left panel) were collected with a fluorescence-activated cell sorter, cultured for 5 d and then stained with biotin-PEG and Alexa Fluor 647-streptavidin (right panel).

Article Snippet: Fifty μg of total protein from the clarified lysate were analyzed by 12.5% reducing SDS-PAGE, transferred to nitrocellulose paper and sequentially stained with biotinylated goat anti-HA antibody against the HA epitope tag sequence YPYDVPDYA (Vector Laboratories) or rabbit anti-tubulin α antibody (NeoMarkers) followed by streptavidin-HRP and goat anti-rabbit Ig-HRP, respectively (Jackson ImmunoResearch Laboratories).

Techniques: Flow Cytometry, Staining, Functional Assay, Binding Assay, Fluorescence, Cell Culture

Journal: mAbs

Article Title: Mimicking the germinal center reaction in hybridoma cells to isolate temperature-selective anti-PEG antibodies

doi: 10.4161/mabs.29124

Figure Lengend Snippet: Figure 7. 2B5 can bind to crown ether structure. ( A ) Illustration of the chemical structures of 18-crown-6, PEG400 and 2-aminomethyl-18-crown-6. ( B ) Graded concentrations of 18-crown-6 were mixed at 4°C with 10 μg/mL 3.3 ( ○ ), 2B5 (■) or 7G8 (●) antibodies prior to addition to 96-well plates coated with amino-PEG 2k -NH 2 (upper panel) or β-glucuronidase (lower panel). Results show antibody binding as a percentage of maximum binding activity. (n = 3). Bars, SD ( C ) Graded concentrations of purified 3.3 ( ○ ), 2B5 (■) or 7G8 (●) antibodies were incubated at 4°C, 25°C or 37°C in microplate wells coated with 2-aminomethyl-18-crown-6. After 4 h, the wells were washed and antibody binding was determined by adding biotin-conjugated goat anti-mouse IgG Fc antibodies and streptavidin-biotinylated peroxidase complex followed by ABTS substrate. The mean absorbance values (405 nm) of triplicate determinations are shown. Bars, SD.

Article Snippet: Fifty μg of total protein from the clarified lysate were analyzed by 12.5% reducing SDS-PAGE, transferred to nitrocellulose paper and sequentially stained with biotinylated goat anti-HA antibody against the HA epitope tag sequence YPYDVPDYA (Vector Laboratories) or rabbit anti-tubulin α antibody (NeoMarkers) followed by streptavidin-HRP and goat anti-rabbit Ig-HRP, respectively (Jackson ImmunoResearch Laboratories).

Techniques: Binding Assay, Activity Assay, Purification, Incubation

Journal: Cell

Article Title: Structural basis for IL-12 and IL-23 receptor sharing reveals a gateway for shaping actions on T versus NK cells.

doi: 10.1016/j.cell.2021.01.018

Figure Lengend Snippet: Figure 3. Affinity maturation of IL-12Rb1 facilitates structural characterization of IL-12 and IL-23 receptor complexes by cryo-EM (A) Schematic of IL-12Rb1 affinity maturation strategy. The D1–D2 domains of IL-12Rb1 were expressed as a fusion with N-terminal Aga2, and an error-prone library was generated. Yeast were selected with biotinylated IL-23, increasing the stringency of selection each round. (B) Affinity maturation of IL-12Rb1 for IL-23 enhances binding to IL-23 and IL-12. Flow cytometry plots showing binding of 10 nM IL-23 (top) or 10 nM IL-12 (bottom) to IL-12Rb1-expressing yeast. Yeast were stained with amyc-AF488 (x axis) as a measure of IL-12Rb1 surface expression and biotinylated cytokine followed by streptavidin-AF647 (y axis) as a measure of cytokine binding. The left-hand column shows isogenic yeast expressing wild-type IL-12Rb1, and subsequent columns show the error-prone library prior to selection (naive) or at different rounds, as indicated. (C) Affinity-enhancing mutations map to the interface between IL-12Rb1 and p40. Interface view of IL-12Rb1 binding to p40 with labels indicating convergent mutations identified in the error-prone selections (Tyr109Ser Gln132Leu). (D) IL-12Rb1 mutations enhance p40 binding in solution. SPR curves showing binding of IL-12Rb1 D1–D2 to immobilized p40. Dissociation constant (KD) was determined using a steady-state affinity model. RU, response units. (E and F) Cryo-EM map of the IL-23 receptor complex as viewed from the top (E) and side (F). Individual subunits are colored as in Figure 1. (G) Cartoon model showing individual components IL-23R (PDB: 5MZV), IL-23 (PDB: 3DUH), and IL-12Rb1 (reported here) fit to the map density.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Sodium Pyruvate GIBCO Cat#11360-070 1M HEPES GIBCO Cat#15630-080 Penicillin-streptomycin GIBCO Cat#15140163 2-mercaptoethanol Sigma Cat#M3148 TrypLE GIBCO Cat#12604013 Carboxypeptidase A Sigma Cat#C9268 Carboxypeptidase B Sigma Cat#217356 Expi293 Expression Medium GIBCO Cat#A1435101 FreeStyle 293 Expression Media GIBCO Cat#12338-026 Dulbecco’s Modified Eagle’s Medium (DMEM) GIBCO Cat#11960-069 Phosphate Buffered Saline (PBS) GIBCO Cat#20012-050 Fetal Bovine Serum Sigma Cat#F4135-500 ACK lysis buffer GIBCO Cat#A10492-01 Sf-900 III Media Invitrogen Cat#12658019 ESF 921 Delta Series Insect Cell Culture Medium, Methionine Deficient Expression Systems Cat#96-200 Selenomethionine Fisher Scientific Cat#AC259960010 ESF 921 Insect Cell Culture Medium Expression Systems Cat#96-001-01 D-glucose (dextrose) Sigma Cat#G8270-5KG YPD broth Sigma Cat#Y1375 Bacto Casamino Acids GIBCO Cat#223050 Yeast Nitrogen Base (without amino acids) RPI Cat#Y20040 Sodium Citrate Tribasic Dihydrate Sigma Cat#S4641 Citric Acid Monohydrate Sigma Cat#C1909 D-galactose Sigma Cat#G0625 Bicine Sigma Cat#B8660-100G Adenosine 50-triphosphate disodium salt hydrate Sigma Cat#A2383 Magnesium Acetate Sigma Cat#M5661-250G Biotin Sigma Cat#B4501 DMSO Fisher Scientific Cat#BP231-100 16% paraformaldehyde Fisher Scientific Cat#50-980-487 Critical commercial assays SA sensor chip Cytiva Cat#BR-1005-31 HBS-P Cytiva Cat#BR-1006-71 SepMate-50 columns StemCell Technologies Cat#85450 GeneMorph II Random Mutagenesis kit Agilent Technologies Cat#200550 Streptavidin microbeads Miltenyi Cat#130-048-101 Superdex 200 Increase column Cytiva Cat#28990944 LS magnetic selection column Miltenyi Cat#130-042-401 EasySep Magnet StemCell Technologies Cat#18000 Zymoprep Yeast Plasmid Miniprep II Kit Zymo Research Cat#D2004 Quantifoil Au1.2/1.3, 200 mesh grids SPI supplies Cat#4220G-XA Mouse NK cell Isolation Kit Miltenyi Cat#130-115-818 Mouse naive CD4+ T cell Isolation Kit Miltenyi Cat#130-104-453 Human CD8+ T cell Isolation Kit Miltenyi Cat#130-096-495 Mouse CD8a+ T cell isolation kit Miltenyi Cat#130-104-075 EasySep Human NK cell Isolation kit StemCell Technologies Cat#17955 (Continued on next page) ll e3 Cell 184, 983–999.e1–e12, February 18, 2021 Article

Techniques: Cryo-EM Sample Prep, Generated, Selection, Binding Assay, Flow Cytometry, Expressing, Staining

Journal: Cell

Article Title: Structural basis for IL-12 and IL-23 receptor sharing reveals a gateway for shaping actions on T versus NK cells.

doi: 10.1016/j.cell.2021.01.018

Figure Lengend Snippet: Figure 5. IL-12 partial agonists preferentially support T cell function with reduced activity on NK cells based on differences in IL-12Rb1 expression (A) Cell-type- and activation-state-dependent expression of IL-12Rb1. Flow cytometry plots showing IL-12Rb1 expression levels measured by mouse p40 tetramer staining of murine NK cells (CD3-NK1.1+) or CD8+ T cells (CD3+CD8+). Red line indicates 200 nM tetramer staining; gray population represents streptavidin staining alone. Single-cell suspension of spleen and lymph nodes from female C57BL/6J mice were stained with p40 tetramer either directly (ex vivo) or following 2-day stimulation with 2.5 mg/mL aCD3, 5 mg/mL aCD28, and 100 IU/mL IL-2 (blasts). For gating, see Figure S4A.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Sodium Pyruvate GIBCO Cat#11360-070 1M HEPES GIBCO Cat#15630-080 Penicillin-streptomycin GIBCO Cat#15140163 2-mercaptoethanol Sigma Cat#M3148 TrypLE GIBCO Cat#12604013 Carboxypeptidase A Sigma Cat#C9268 Carboxypeptidase B Sigma Cat#217356 Expi293 Expression Medium GIBCO Cat#A1435101 FreeStyle 293 Expression Media GIBCO Cat#12338-026 Dulbecco’s Modified Eagle’s Medium (DMEM) GIBCO Cat#11960-069 Phosphate Buffered Saline (PBS) GIBCO Cat#20012-050 Fetal Bovine Serum Sigma Cat#F4135-500 ACK lysis buffer GIBCO Cat#A10492-01 Sf-900 III Media Invitrogen Cat#12658019 ESF 921 Delta Series Insect Cell Culture Medium, Methionine Deficient Expression Systems Cat#96-200 Selenomethionine Fisher Scientific Cat#AC259960010 ESF 921 Insect Cell Culture Medium Expression Systems Cat#96-001-01 D-glucose (dextrose) Sigma Cat#G8270-5KG YPD broth Sigma Cat#Y1375 Bacto Casamino Acids GIBCO Cat#223050 Yeast Nitrogen Base (without amino acids) RPI Cat#Y20040 Sodium Citrate Tribasic Dihydrate Sigma Cat#S4641 Citric Acid Monohydrate Sigma Cat#C1909 D-galactose Sigma Cat#G0625 Bicine Sigma Cat#B8660-100G Adenosine 50-triphosphate disodium salt hydrate Sigma Cat#A2383 Magnesium Acetate Sigma Cat#M5661-250G Biotin Sigma Cat#B4501 DMSO Fisher Scientific Cat#BP231-100 16% paraformaldehyde Fisher Scientific Cat#50-980-487 Critical commercial assays SA sensor chip Cytiva Cat#BR-1005-31 HBS-P Cytiva Cat#BR-1006-71 SepMate-50 columns StemCell Technologies Cat#85450 GeneMorph II Random Mutagenesis kit Agilent Technologies Cat#200550 Streptavidin microbeads Miltenyi Cat#130-048-101 Superdex 200 Increase column Cytiva Cat#28990944 LS magnetic selection column Miltenyi Cat#130-042-401 EasySep Magnet StemCell Technologies Cat#18000 Zymoprep Yeast Plasmid Miniprep II Kit Zymo Research Cat#D2004 Quantifoil Au1.2/1.3, 200 mesh grids SPI supplies Cat#4220G-XA Mouse NK cell Isolation Kit Miltenyi Cat#130-115-818 Mouse naive CD4+ T cell Isolation Kit Miltenyi Cat#130-104-453 Human CD8+ T cell Isolation Kit Miltenyi Cat#130-096-495 Mouse CD8a+ T cell isolation kit Miltenyi Cat#130-104-075 EasySep Human NK cell Isolation kit StemCell Technologies Cat#17955 (Continued on next page) ll e3 Cell 184, 983–999.e1–e12, February 18, 2021 Article

Techniques: Cell Function Assay, Activity Assay, Expressing, Activation Assay, Flow Cytometry, Staining, Suspension, Ex Vivo

Journal: Nature immunology

Article Title: Unique pattern of neutrophil migration and function during tumor progression

doi: 10.1038/s41590-018-0229-5

Figure Lengend Snippet: a . Transwell assays evaluating the ability of BM neutrophils from mice bearing s.c. 4662 tumors derived from KPC mice to spontaneously migrate (left) and chemotaxis in response to CXCL1 (middle) or to fMPL (right). Mean, and SD are shown (n=4). P values are calculated in two-sided Student’s t-test (not shown due to lack of significance). b . Transwell assays evaluating the ability of neutrophils to spontaneously migrate. In one week LL2 experiments, n=5 for control group and n=6 for LL2 mice. In three week LL2 experiments, n=3 for control mice and n=4 for LL2 mice. Mean, and SD are shown. c . Transwell assays of neutrophil chemotaxis in response to CXCL1. Mean and SD are shown. (d, e) Flow cytometry analysis of CD45.2 + BM neutrophils from control mice and CD45.1 + neutrophils from one-week or three-week LL2 TB mice to migrate into the spleen and lung 1 hour after injection to naive CD45.1 + CD45.2 + recipients. d . Flow cytometry gating strategy with representative result from 6 performed experiments, e . Ratio between CD45.1 + and CD45.2 + CD11b + Ly6G + Ly6C lo neutrophils. In each experiment, the ratio of CD45.1 + /CD45.2 + cells injected into recipient mice was set as the baseline= 1. For one week LL2 experiments n=8, for three-week LL2 experiments n=6. Mean, and SD are shown. P values are calculated in two-sided Student’s t-test from baseline within each group of LL2 mice and between the two groups of LL2 mice are shown. f . Transwell migration assay of BM neutrophils from control and KPC mice with PanIn and invasive PDA. N=5 (control), n=5 (PanIn), n=3 (PDA). Mean and SD are shown. g . The mean-squared displacements of neutrophil migration. Each curve represents cumulative results of traces 45–60 individual cells. In a–c, e,f p values were calculated in two-sided Student’s t-test. * − p <0.05, **−p<0.01. In g . p values were calculated in two-way ANOVA test with Bonferroni correction for multiple comparisons. ***−p<0.0001 Two experiments with similar results were performed.

Article Snippet: For BM Ly6G + cells isolation, cells were labeled with biotinylated anti-Ly6G antibody (Miltenyi Biotec), incubated with streptavidin-coated microbeads (Miltenyi Biotec) and separated on MACS columns (Miltenyi Biotec).

Techniques: Derivative Assay, Chemotaxis Assay, Control, Flow Cytometry, Injection, Transwell Migration Assay, Migration

Journal: Nature immunology

Article Title: Unique pattern of neutrophil migration and function during tumor progression

doi: 10.1038/s41590-018-0229-5

Figure Lengend Snippet: a . Antigen-specific proliferation of CD8 + T cells in the presence of BM Ly6G + cells isolated from control mice and one or three weeks LL2 mice. Transgenic, Pmel splenocytes stimulated with specific peptide were used as responders. Proliferation was measured in triplicate by 3 H thymidine uptake. Baseline of T cell proliferation in the absence of added neutrophils was set as 100%. Control -n=7, week 1 LL2 - n=10, week 3 LL2 - n=11. Mean, and SD are shown. b . Antigen-specific proliferation of CD8 + T cells in the presence of BM Ly6G + cells from naive mice, RET melanoma mice (left panel), LLC, or EL4 (right panel) mice. Pmel splenocytes stimulated with specific peptide were used as responders. Proliferation was measured in triplicate by 3 H thymidine uptake. One experiment is shown. Three experiments with the same results were performed. Each experiment included 3 mice. Mean and SD are shown. c . Suppressive activity of PMN from BM of KPC and TRAMP transgenic mice. OT-1 splenocytes stimulated with specific peptide were used as responders. Proliferation was measured in triplicate by 3 H thymidine uptake. For KPC mice 5 experiments with similar results were performed. In TRAMP mice two experiments with the same results were performed. Mean and SD are shown. d . Suppressive activity of neutrophils from KPC mice with PanIN and PDA. Proliferation was evaluated by 3 H-thymidine uptake in triplicates. Means and SD of cumulative result from two mice are shown. e . Cytokine protein expression array in sera of one- and three-week orthotopic LL2 TB mice. Experiments were performed in duplicates and individual data are shown. On the left cytokines with more than 2SD increase in sera of three-week LL2 mice over one-week LL2 mice. On the right cytokines with less than 2SD increase or decreased in three-week LL2 mice. * − p <0.05; **−p<0.01 in two-sided Student’s t-test.

Article Snippet: For BM Ly6G + cells isolation, cells were labeled with biotinylated anti-Ly6G antibody (Miltenyi Biotec), incubated with streptavidin-coated microbeads (Miltenyi Biotec) and separated on MACS columns (Miltenyi Biotec).

Techniques: Isolation, Control, Transgenic Assay, Activity Assay, Expressing

Journal: Nature immunology

Article Title: Unique pattern of neutrophil migration and function during tumor progression

doi: 10.1038/s41590-018-0229-5

Figure Lengend Snippet: Spontaneous (n=24 for both healthy donors and cancer patients groups) ( a ) or chemokine induced (for healthy donor n=24 for media, n=10 for CXCL8 stimulation, n=6 for fMLP stimulation; for cancer patients n= 14 for media, n=3 for CXCL8, and n=3 for fMLP); ( b) migration of neutrophils from healthy individuals and cancer subjects. Individual results for each subject, mean and SD are shown. c . CXCR1 and CXCR2 expression in neutrophils from healthy individual (n=7) and cancer patients (n=4 for CXCR1 and n=6 for CXCR2) assessed by flow cytometry. Individual results for each subject, mean and SD are shown. d, e . Spontaneous (n=12) (d) and CXCL1 stimulated (n=11 for PMN and n=13 for PMN-MDSC) ( e ) migration of neutrophils and PMN-MDSC from the same cancer patients. f . Expression of chemokine receptors in human neutrophils. Indicated chemokine receptors were measured by flow cytometry on PMN-MDSC and neutrophils from cancer patients. N=4 for CXCR1 and n=6 for CXCR2. g . Percentage of CD45 − CFSE + LLC cells in lungs from naive mice 12 hours after intravenous injection. BM Ly6G + neutrophils (PMN) from naïve (n=7), one week (n=5), or three-week (n=5) LL2 mice were injected 6 hours before tumor cells. h . IVIS-based analysis of ex-vivo luciferase activity in excised lungs from naive mice, which were injected i.v. with indicated BM Ly6G + cells 5 hours before the i.v. injection of LL2 tumor cells. N=3. Detection was performed two weeks after the injection. In all panels mean and SD are shown. P values are calculated in two-sided Student’s t-test. * − p <0.05; **−p<0.01; ***−p<0.001; ****−p<0.0001.

Article Snippet: For BM Ly6G + cells isolation, cells were labeled with biotinylated anti-Ly6G antibody (Miltenyi Biotec), incubated with streptavidin-coated microbeads (Miltenyi Biotec) and separated on MACS columns (Miltenyi Biotec).

Techniques: Migration, Expressing, Flow Cytometry, Injection, Ex Vivo, Luciferase, Activity Assay

Journal: The Journal of experimental medicine

Article Title: A dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN)-related protein is highly expressed on human liver sinusoidal endothelial cells and promotes HIV-1 infection.

doi: 10.1084/jem.193.6.671

Figure Lengend Snippet: Figure 4. L-SIGN binds ICAM-3 (A) and HIV-1 gp120 (B). Adhesion of ICAM-3 and gp120 to the K562–L-SIGN and K562–DC-SIGN cells was measured with the fluorescent bead adhesion assay (reference 11). The y-axis represents the percent- age of cells binding ligand-coated fluorescent beads. The L-SIGN cross-reacting mAbs AZN-D2 (20 mg/ml) and AZN-D3 (20 mg/ml) inhibit the adhesion of ICAM-3 and gp120 to L-SIGN, in contrast to the DC-SIGN–specific mAb AZN-D1 (20 mg/ml). Adhesion of both ICAM-3 and gp120 to the K562 transfectants is also inhibited by either 20 mg/ml mannan or 5 mM EGTA. Adhesion of both ligands to mock transfectants was ,5%. One representative experiment out of three is shown (SD , 5%).

Article Snippet: Fluorescent beads were coated with M-tropic HIV-1 MN envelope glycoprotein gp120 as follows: streptavidin-coated fluorescent beads were incubated with biotinylated F(ab 9 ) 2 fragment rabbit anti–sheep IgG (6 m g/ml; Jackson ImmunoResearch Laboratories) followed by an overnight incubation with sheep antigp120 antibody D7324 (Alto Bio Reagents, Ltd.) at 4 8 C. The beads were washed and incubated with 250 ng/ml purified HIV-1 gp120 (provided by Immunodiagnostics, Inc., through the National Institutes of Health AIDS Research and Reference Reagent Program) overnight at 4 8 C. The fluorescent beads adhesion assay was performed as described by Geijtenbeek et al. (11).

Techniques: Cell Adhesion Assay, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Tumor-Specific miRNA Signatures in Combination with CA19-9 for Liquid Biopsy-Based Detection of PDAC

doi: 10.3390/ijms222413621

Figure Lengend Snippet: Enrichment of pancreatic tumor cell-derived EVs by immunoprecipitation with magnetic beads. ( A ) Particle size distribution of EVs isolated from HPNE, AsPC−1, BxPC−3, Mia PaCa−2, and PANC−1 cell lines. The area under the curve represents the absolute number of particles isolated from the concentrated culture medium. ( B ) Mean particle sizes of EVs were analyzed in triplicate and graphed. ( C ) SEM images of EVs bound to surfaces of magnetic microbeads after immunoaffinity capture. Microbeads were functionalized with antibodies against the pancreatic cancer associated surface markers ITGAV, ITGA2, and GPC1. Scale bars represent 200 nm.

Article Snippet: The surface profiles of cells and EVs bound to 15 μm microbeads (SVP 150-4, SPHERO™ Streptavidin Coated Particles, Spherotech Inc., Lake Forest, IL, USA) coated with antibodies targeting tumor-associated surface markers were analyzed via flow cytometry.

Techniques: Derivative Assay, Immunoprecipitation, Magnetic Beads, Isolation