anti igg  (Valiant)


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    Name:
    IgG f ab 2 human purified
    Description:
    Product is the lyophilized powder of human IgG F ab 2 and buffer salts
    Catalog Number:
    0855910
    Price:
    356.6
    Applications:
    Immunoassays
    Size:
    10 mg
    Category:
    Life Sciences Biochemicals Proteins and Derivatives Tissue and Blood Products
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    Structured Review

    Valiant anti igg
    IgG f ab 2 human purified
    Product is the lyophilized powder of human IgG F ab 2 and buffer salts
    https://www.bioz.com/result/anti igg/product/Valiant
    Average 93 stars, based on 35 article reviews
    Price from $9.99 to $1999.99
    anti igg - by Bioz Stars, 2021-02
    93/100 stars

    Images

    1) Product Images from "Interferon regulatory factor 8 regulates caspase-1 expression to facilitate Epstein-Barr virus reactivation in response to B cell receptor stimulation and chemical induction"

    Article Title: Interferon regulatory factor 8 regulates caspase-1 expression to facilitate Epstein-Barr virus reactivation in response to B cell receptor stimulation and chemical induction

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006868

    IRF8 depletion suppresses caspase activation and caspase activation is required for EBV lytic replication. A. IRF8 depletion suppresses caspase activation. Western blot analysis of protein extracts from Fig 1D using antibodies against caspase-3, cleaved caspase-3, caspase-8, cleaved caspase-8, caspase-7, cleaved caspase-7, caspase-9, cleaved caspass-9, caspase-2 and Bcl-2 as indicated. B. Caspase inhibition suppresses EBV lytic gene expression. Akata (EBV + ) cells were untreated or pre-treated with pan-caspase inhibitor (Z-VAD-FMK) for 1 hr and then anti-IgG was added for 48 hrs. RNA was extracted and EBV lytic gene expression was analyzed by RT-qPCR. Data are presented as means ± standard deviations of triplicate assays. ** p
    Figure Legend Snippet: IRF8 depletion suppresses caspase activation and caspase activation is required for EBV lytic replication. A. IRF8 depletion suppresses caspase activation. Western blot analysis of protein extracts from Fig 1D using antibodies against caspase-3, cleaved caspase-3, caspase-8, cleaved caspase-8, caspase-7, cleaved caspase-7, caspase-9, cleaved caspass-9, caspase-2 and Bcl-2 as indicated. B. Caspase inhibition suppresses EBV lytic gene expression. Akata (EBV + ) cells were untreated or pre-treated with pan-caspase inhibitor (Z-VAD-FMK) for 1 hr and then anti-IgG was added for 48 hrs. RNA was extracted and EBV lytic gene expression was analyzed by RT-qPCR. Data are presented as means ± standard deviations of triplicate assays. ** p

    Techniques Used: Activation Assay, Western Blot, Inhibition, Expressing, Quantitative RT-PCR

    IRF8 depletion suppresses the expression of genes involved in apoptosis. A. Schematic representation of RNA-seq analyses of Akata (EBV + ) cells carrying control (NC) or IRF8-sg2 sgRNAs, RNAs were extracted from cells derived from three distinct lentiviral transductions. Using 2-fold change as a cutoff, 196 and 57 genes were down- or up-regulated upon IRF8 depletion, respectively. Gene Ontology analysis showing that 19 genes involved in “positive regulation of apoptosis” (red dots) were down-regulated by IRF8 depletion. B. Fold changes of the 19 apoptosis-related genes and the validation of 8 of them by RT-qPCR analysis of RNAs from cells derived from three distinct lentiviral transductions. C. IRF8 depletion (sg2) suppresses caspase-1 expression and the generation of cleaved caspase substrates upon lytic induction by anti-IgG cross-linking. Western blot analysis of protein extracts from Fig 1D using antibodies against caspase-1, PARP, and cleaved caspase substrates (Peptides containing [DE(T/S/A)D] motif) as indicated.
    Figure Legend Snippet: IRF8 depletion suppresses the expression of genes involved in apoptosis. A. Schematic representation of RNA-seq analyses of Akata (EBV + ) cells carrying control (NC) or IRF8-sg2 sgRNAs, RNAs were extracted from cells derived from three distinct lentiviral transductions. Using 2-fold change as a cutoff, 196 and 57 genes were down- or up-regulated upon IRF8 depletion, respectively. Gene Ontology analysis showing that 19 genes involved in “positive regulation of apoptosis” (red dots) were down-regulated by IRF8 depletion. B. Fold changes of the 19 apoptosis-related genes and the validation of 8 of them by RT-qPCR analysis of RNAs from cells derived from three distinct lentiviral transductions. C. IRF8 depletion (sg2) suppresses caspase-1 expression and the generation of cleaved caspase substrates upon lytic induction by anti-IgG cross-linking. Western blot analysis of protein extracts from Fig 1D using antibodies against caspase-1, PARP, and cleaved caspase substrates (Peptides containing [DE(T/S/A)D] motif) as indicated.

    Techniques Used: Expressing, RNA Sequencing Assay, Derivative Assay, Quantitative RT-PCR, Western Blot

    2) Product Images from "Mucosal IgA Responses in Healthy Adult Volunteers following Intranasal Spray Delivery of a Live Attenuated Measles Vaccine ▿"

    Article Title: Mucosal IgA Responses in Healthy Adult Volunteers following Intranasal Spray Delivery of a Live Attenuated Measles Vaccine ▿

    Journal: Clinical and Vaccine Immunology : CVI

    doi: 10.1128/CVI.00354-10

    Total IgG and IgA measured in oral fluid and nasal washes. Nonimmune (A) and immune (B) volunteers were vaccinated as described in the legend to Fig. . Total IgG and sIgA were measured in oral fluid and nasal washes and reported in μg/ml.
    Figure Legend Snippet: Total IgG and IgA measured in oral fluid and nasal washes. Nonimmune (A) and immune (B) volunteers were vaccinated as described in the legend to Fig. . Total IgG and sIgA were measured in oral fluid and nasal washes and reported in μg/ml.

    Techniques Used:

    Correlations between serum PRN and MV-specific IgG and IgA titers in serum, oral fluid, and nasal washes for nonimmune and immune subjects vaccinated i.n. and s.c. The graphics show correlations between peak serum PRN (day 14) and MV-specific peak IgG
    Figure Legend Snippet: Correlations between serum PRN and MV-specific IgG and IgA titers in serum, oral fluid, and nasal washes for nonimmune and immune subjects vaccinated i.n. and s.c. The graphics show correlations between peak serum PRN (day 14) and MV-specific peak IgG

    Techniques Used:

    3) Product Images from "Caspases switch off m6A RNA modification pathway to reactivate a ubiquitous human tumor virus"

    Article Title: Caspases switch off m6A RNA modification pathway to reactivate a ubiquitous human tumor virus

    Journal: bioRxiv

    doi: 10.1101/2020.11.12.377127

    See also Figures 6 , 7 and Table S2. (A) V5-METTL3 was incubated with individual caspase for 2 hrs at 37°C. Western Blot was performed using anti-METTL3 and anti-V5 antibodies as indicated. The locations of antibody recognition epitopes were labelled as indicated. The positions of weakly cleaved fragments were labelled by arrowhead. Star denotes non-specific bands. (B) V5-tagged WTAP D301A/D302A and D301A mutants were incubated with individual recombinant caspase for 2 hrs. Western Blot was performed using antibodies as indicated. Arrowheads denote cleaved fragments. (C) Sequence alignment of WTAP sequences from 10 representative species using the Constraint-based Multiple Alignment Tool (COBALT). The cleavage motifs were highlighted by yellow color. (D) Motif analysis showing the conservation of the WTAP D302 and the surrounding amino acids. Amino acid sequences were extracted from 97 vertebrate species and motif logos were generated using WebLogo. (E-F) Akata (EBV+) cells were used to establish stable cell lines using 2 different guide RNA constructs targeting YTHDF1 (D) and ALKBH5 (E) and a non-targeting control (sg-NC). The cells were untreated or lytically induced with anti-IgG-mediated BCR activation. Cellular and viral protein expression levels were monitored by Western Blot using antibodies as indicated.
    Figure Legend Snippet: See also Figures 6 , 7 and Table S2. (A) V5-METTL3 was incubated with individual caspase for 2 hrs at 37°C. Western Blot was performed using anti-METTL3 and anti-V5 antibodies as indicated. The locations of antibody recognition epitopes were labelled as indicated. The positions of weakly cleaved fragments were labelled by arrowhead. Star denotes non-specific bands. (B) V5-tagged WTAP D301A/D302A and D301A mutants were incubated with individual recombinant caspase for 2 hrs. Western Blot was performed using antibodies as indicated. Arrowheads denote cleaved fragments. (C) Sequence alignment of WTAP sequences from 10 representative species using the Constraint-based Multiple Alignment Tool (COBALT). The cleavage motifs were highlighted by yellow color. (D) Motif analysis showing the conservation of the WTAP D302 and the surrounding amino acids. Amino acid sequences were extracted from 97 vertebrate species and motif logos were generated using WebLogo. (E-F) Akata (EBV+) cells were used to establish stable cell lines using 2 different guide RNA constructs targeting YTHDF1 (D) and ALKBH5 (E) and a non-targeting control (sg-NC). The cells were untreated or lytically induced with anti-IgG-mediated BCR activation. Cellular and viral protein expression levels were monitored by Western Blot using antibodies as indicated.

    Techniques Used: Incubation, Western Blot, Recombinant, Sequencing, Generated, Stable Transfection, Construct, Activation Assay, Expressing

    YTHDF2 is cleaved by caspases in vivo and in vitro . (A) Western Blot showing YTHDF2 downregulation by IgG cross-linking induced BCR activation. Akata (EBV+) and Akata-4E3 (EBV-) cells were treated with anti-IgG antibody as indicated. YTHDF2 and viral protein expression levels were monitored by Western Blot. Arrowheads denote cleaved YTHDF2 in the longer exposure blot. (B) Caspase inhibition blocks YTHDF2 degradation. The cells were either untreated or pretreated with a pan-caspase inhibitor (Z-VAD-FMK, 50 μM) for 1 hr, and then anti-IgG antibody was added for 48 hrs. Arrowheads denote cleaved YTHDF2. (C) Functional domains and putative cleavage sites in YTHDF2. CaspDB was used to predict the potential cleavage sites in YTHDF2. The locations of the putative cleavage sites D166 and D367 were labeled as indicated. CNOT1 binding domain: responsible for the degradation of associated RNA; P/Q/N rich region: aggregation-prone region; YTH domain: responsible for binding to m 6 A-modified RNA. (D) Schematic representation of V5-tagged YTHDF2 with two putative cleavage sites. Red oval, anti-YTHDF2 monoclonal antibody recognition site. (E-F). Wild-type V5-YTHDF2 was incubated with individual recombinant caspase for 2 hrs. Western Blot was performed using either anti-YTHDF2 (E) or anti-V5 (F) antibodies. The relative position of predicted cleavage fragments was labeled as indicated. (G-H) YTHDF2 (D166A/D367A) mutant protein was incubated with individual recombinant caspase for 2 hrs. Western Blot was performed using antibodies as indicated. (I) Motif analysis showing the conservation of the two cleavage sites and the surrounding amino acids. Amino acid sequences were extracted from 97 (D166) and 80 (D367) vertebrate species and motif logos were generated using WebLogo. (J) Structure modeling of full-length YTHDF2 by I-TASSER. The two cleavage sites D166 and D367 are labeled as indicated. N and C denote N-terminus and C-terminus, respectively. (K) Triple depletion of caspase-3, −8 and −6 reduces YTHDF2 and PIAS1 degradation and blocks viral protein accumulation. The CASP3/CASP8/CASP6-triply-depleted Akata (EBV+) cells were lytically induced by anti-IgG treatment. The expression of cleaved caspases, YTHDF2, PIAS1 and viral proteins was monitored by Western Blot using antibodies as indicated. See also Figure S2 and Table S2.
    Figure Legend Snippet: YTHDF2 is cleaved by caspases in vivo and in vitro . (A) Western Blot showing YTHDF2 downregulation by IgG cross-linking induced BCR activation. Akata (EBV+) and Akata-4E3 (EBV-) cells were treated with anti-IgG antibody as indicated. YTHDF2 and viral protein expression levels were monitored by Western Blot. Arrowheads denote cleaved YTHDF2 in the longer exposure blot. (B) Caspase inhibition blocks YTHDF2 degradation. The cells were either untreated or pretreated with a pan-caspase inhibitor (Z-VAD-FMK, 50 μM) for 1 hr, and then anti-IgG antibody was added for 48 hrs. Arrowheads denote cleaved YTHDF2. (C) Functional domains and putative cleavage sites in YTHDF2. CaspDB was used to predict the potential cleavage sites in YTHDF2. The locations of the putative cleavage sites D166 and D367 were labeled as indicated. CNOT1 binding domain: responsible for the degradation of associated RNA; P/Q/N rich region: aggregation-prone region; YTH domain: responsible for binding to m 6 A-modified RNA. (D) Schematic representation of V5-tagged YTHDF2 with two putative cleavage sites. Red oval, anti-YTHDF2 monoclonal antibody recognition site. (E-F). Wild-type V5-YTHDF2 was incubated with individual recombinant caspase for 2 hrs. Western Blot was performed using either anti-YTHDF2 (E) or anti-V5 (F) antibodies. The relative position of predicted cleavage fragments was labeled as indicated. (G-H) YTHDF2 (D166A/D367A) mutant protein was incubated with individual recombinant caspase for 2 hrs. Western Blot was performed using antibodies as indicated. (I) Motif analysis showing the conservation of the two cleavage sites and the surrounding amino acids. Amino acid sequences were extracted from 97 (D166) and 80 (D367) vertebrate species and motif logos were generated using WebLogo. (J) Structure modeling of full-length YTHDF2 by I-TASSER. The two cleavage sites D166 and D367 are labeled as indicated. N and C denote N-terminus and C-terminus, respectively. (K) Triple depletion of caspase-3, −8 and −6 reduces YTHDF2 and PIAS1 degradation and blocks viral protein accumulation. The CASP3/CASP8/CASP6-triply-depleted Akata (EBV+) cells were lytically induced by anti-IgG treatment. The expression of cleaved caspases, YTHDF2, PIAS1 and viral proteins was monitored by Western Blot using antibodies as indicated. See also Figure S2 and Table S2.

    Techniques Used: In Vivo, In Vitro, Western Blot, Activation Assay, Expressing, Inhibition, Functional Assay, Labeling, Binding Assay, Modification, Incubation, Recombinant, Mutagenesis, Generated

    Identification of new potential caspase substrates during EBV reactivation. (A) The cleavage motifs derived from PIAS1 (LTYD*G and NGVD*G) were used to virtually screen the entire human proteome for proteins sharing the same sequences. The human proteome dataset containing approximately 20,000 human protein-coding genes represented by the canonical protein sequence was downloaded from UniProtKB/Swiss-Prot. (B) 16 additional proteins were extracted from the screen. 8 proteins carry the LTYD*G motif (left) and 8 proteins carry the NGVD*G motif (right). 6 proteins (underlined) were selected for further validation. (C) Protein downregulation during EBV reactivation. Akata (EBV+) cells was treated with anti-IgG antibody to induce EBV reactivation for 0, 24 and 48 hrs. Western Blot showing the downregulation of 6 selected proteins using antibodies as indicated. SAMHD1 and β-actin were included as controls. Arrowhead denotes the cleaved fragment for EHMT2. (D) Caspase inhibition blocks the degradation of YTHDF2, MAGEA10, SORT1 MTA1 and EHMT2. The Akata (EBV+) cells were either untreated or pretreated with a caspase-3/-7 inhibitor (Z-DEVD-FMK, 50 μM) or pan-caspase inhibitor (Z-VAD-FMK, 50 μM) for 1 hr, and then anti-IgG antibody was added for 48 hrs. Western Blot showing the protein levels of 6 selected proteins using antibodies as indicated. SAMHD1 and β-actin were included as controls. Arrowhead denotes cleaved EHMT2 fragment.
    Figure Legend Snippet: Identification of new potential caspase substrates during EBV reactivation. (A) The cleavage motifs derived from PIAS1 (LTYD*G and NGVD*G) were used to virtually screen the entire human proteome for proteins sharing the same sequences. The human proteome dataset containing approximately 20,000 human protein-coding genes represented by the canonical protein sequence was downloaded from UniProtKB/Swiss-Prot. (B) 16 additional proteins were extracted from the screen. 8 proteins carry the LTYD*G motif (left) and 8 proteins carry the NGVD*G motif (right). 6 proteins (underlined) were selected for further validation. (C) Protein downregulation during EBV reactivation. Akata (EBV+) cells was treated with anti-IgG antibody to induce EBV reactivation for 0, 24 and 48 hrs. Western Blot showing the downregulation of 6 selected proteins using antibodies as indicated. SAMHD1 and β-actin were included as controls. Arrowhead denotes the cleaved fragment for EHMT2. (D) Caspase inhibition blocks the degradation of YTHDF2, MAGEA10, SORT1 MTA1 and EHMT2. The Akata (EBV+) cells were either untreated or pretreated with a caspase-3/-7 inhibitor (Z-DEVD-FMK, 50 μM) or pan-caspase inhibitor (Z-VAD-FMK, 50 μM) for 1 hr, and then anti-IgG antibody was added for 48 hrs. Western Blot showing the protein levels of 6 selected proteins using antibodies as indicated. SAMHD1 and β-actin were included as controls. Arrowhead denotes cleaved EHMT2 fragment.

    Techniques Used: Derivative Assay, Sequencing, Western Blot, Inhibition

    YTHDF2 regulates CASP8 mRNA stability through m 6 A modifications. (A-B) YTHDF2 depletion promotes CASP8 mRNA expression. Akata (EBV+) cells and P3HR-1 cells carrying different sgRNA targeting YTHDF2 or control (sg-NC) were used to extract total RNA and qPCR analyses were performed a group of YTHDF2-targeted cellular genes involved in caspase activation. The values were normalized with a non YTHDF2 target HPRT1 . The values of sg-NC were set as 1. (C-D) CASP8 is modified by m 6 A and YTHDF2 binding to CASP8 . Akata (EBV+) cells were used to perform m6A RIP-qPCR (C) and YTHDF2 RIP-qPCR (D), respectively. Values are displayed as fold change over 10% input. (E-G) YTHDF2 depletion promotes caspase-8 protein expression and PIAS1 cleavage upon lytic i nduction. Akata (EBV+) cells (E), P3HR-1 cells (F) and SNU-719 cells (G) carrying different sgRNA targeting YTHDF2 or control (sg-NC) were lytically induced by anti-IgG, TPA and sodium butyrate (NaBu) and gemcitabine treatment for 24 hrs. Protein expression was monitored by Western Blot using antibodies as indicated. (H) CASP8 m 6 A peaks were extracted from MeT-DB V2.0 database. YTHDF2-PAR-CLIP data were retrieved from Wang et al.( Wang et al., 2014 ). The Exon-7 of CASP8 with highest m 6 A peaks were analyzed for conservation among sequences derived from 100 vertebrate species. 15 potential m 6 A motifs (M1-M15) were extracted based on m 6 A motif DRACH. (I) Motif logos were generated for 15 individual sites. Red cycles denote highly conserved motifs (M2, M3, M5, M8 and M12) across 100 vertebrate species. (J-K) WT and mutant CASP8 -Exon-7 were cloned into the m 6 A-null Renilla luciferase (RLuc) reporter (3’UTR region) that also express Firefly luciferase (FLuc) from a separate promoter (J). These three reporter plasmids were transfected into parental or YTHDF2-depleted (YTHDF2 KD) SNU719 cells. Relative Renilla to Filefly luciferase activity (RLuc/FLuc) was calculated (K). The value of WT in parental cells was set as 1. (L) Model illustrating YTHDF2 regulation of CASP8 mRNA and caspase-8 regulation of YTHDF2 and PIAS1 in EBV reactivation. Results from three biological replicates are presented. Error bars indicate ±SD. *, p
    Figure Legend Snippet: YTHDF2 regulates CASP8 mRNA stability through m 6 A modifications. (A-B) YTHDF2 depletion promotes CASP8 mRNA expression. Akata (EBV+) cells and P3HR-1 cells carrying different sgRNA targeting YTHDF2 or control (sg-NC) were used to extract total RNA and qPCR analyses were performed a group of YTHDF2-targeted cellular genes involved in caspase activation. The values were normalized with a non YTHDF2 target HPRT1 . The values of sg-NC were set as 1. (C-D) CASP8 is modified by m 6 A and YTHDF2 binding to CASP8 . Akata (EBV+) cells were used to perform m6A RIP-qPCR (C) and YTHDF2 RIP-qPCR (D), respectively. Values are displayed as fold change over 10% input. (E-G) YTHDF2 depletion promotes caspase-8 protein expression and PIAS1 cleavage upon lytic i nduction. Akata (EBV+) cells (E), P3HR-1 cells (F) and SNU-719 cells (G) carrying different sgRNA targeting YTHDF2 or control (sg-NC) were lytically induced by anti-IgG, TPA and sodium butyrate (NaBu) and gemcitabine treatment for 24 hrs. Protein expression was monitored by Western Blot using antibodies as indicated. (H) CASP8 m 6 A peaks were extracted from MeT-DB V2.0 database. YTHDF2-PAR-CLIP data were retrieved from Wang et al.( Wang et al., 2014 ). The Exon-7 of CASP8 with highest m 6 A peaks were analyzed for conservation among sequences derived from 100 vertebrate species. 15 potential m 6 A motifs (M1-M15) were extracted based on m 6 A motif DRACH. (I) Motif logos were generated for 15 individual sites. Red cycles denote highly conserved motifs (M2, M3, M5, M8 and M12) across 100 vertebrate species. (J-K) WT and mutant CASP8 -Exon-7 were cloned into the m 6 A-null Renilla luciferase (RLuc) reporter (3’UTR region) that also express Firefly luciferase (FLuc) from a separate promoter (J). These three reporter plasmids were transfected into parental or YTHDF2-depleted (YTHDF2 KD) SNU719 cells. Relative Renilla to Filefly luciferase activity (RLuc/FLuc) was calculated (K). The value of WT in parental cells was set as 1. (L) Model illustrating YTHDF2 regulation of CASP8 mRNA and caspase-8 regulation of YTHDF2 and PIAS1 in EBV reactivation. Results from three biological replicates are presented. Error bars indicate ±SD. *, p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Activation Assay, Modification, Binding Assay, Western Blot, Cross-linking Immunoprecipitation, Derivative Assay, Generated, Mutagenesis, Clone Assay, Luciferase, Transfection, Activity Assay

    See also Figures 4 and 5 . (A) A group of genes in the category of “ activation of cysteine-type endopeptidase activity involved in apoptotic process ” (also called “ caspase activation ”) were extracted from YTHDF2 target genes derived from YTHDF2 RIP-seq and PAR-CLIP-seq datasets ( Liu et al., 2015 ; Liu et al., 2018 ; Wang et al., 2014 ) (B) YTHDF2 reconstitution suppresses caspase-8 expression and subsequent caspase activation. Akata (EBV+) YTHDF2-sg2 cells were reconstituted with WT or cleavage-resistant YTHDF2 (D166A/D367A) using lentiviral constructs. Western Blot analysis showing the levels for caspase-8 (CASP8), cleaved caspase-8, caspase-3 (CASP3) and cleaved caspase substrates (CASP sub.) in these cell lines upon IgG cross-linking as indicated. (C-D) Caspase-8 inhibition suppress EBV replication in YTHDF2-depleted cells. Control and YTHDF2-depleted Akata (EBV+) cells were either untreated or pretreated with caspase-8 inhibitor (Z-IETD-FMK, 50 μM) for 1 hr and then anti-IgG antibody was added for 0 to 48 hrs. Western Blot showing the protein levels of EBV ZTA and RTA as indicated (C). Extracellular viral DNA was measured by qPCR using primers specific to BALF5 (D). The value of vector control at 0 hr was set as 1. Results from three biological replicates are presented. Error bars indicate ±SD. **, p
    Figure Legend Snippet: See also Figures 4 and 5 . (A) A group of genes in the category of “ activation of cysteine-type endopeptidase activity involved in apoptotic process ” (also called “ caspase activation ”) were extracted from YTHDF2 target genes derived from YTHDF2 RIP-seq and PAR-CLIP-seq datasets ( Liu et al., 2015 ; Liu et al., 2018 ; Wang et al., 2014 ) (B) YTHDF2 reconstitution suppresses caspase-8 expression and subsequent caspase activation. Akata (EBV+) YTHDF2-sg2 cells were reconstituted with WT or cleavage-resistant YTHDF2 (D166A/D367A) using lentiviral constructs. Western Blot analysis showing the levels for caspase-8 (CASP8), cleaved caspase-8, caspase-3 (CASP3) and cleaved caspase substrates (CASP sub.) in these cell lines upon IgG cross-linking as indicated. (C-D) Caspase-8 inhibition suppress EBV replication in YTHDF2-depleted cells. Control and YTHDF2-depleted Akata (EBV+) cells were either untreated or pretreated with caspase-8 inhibitor (Z-IETD-FMK, 50 μM) for 1 hr and then anti-IgG antibody was added for 0 to 48 hrs. Western Blot showing the protein levels of EBV ZTA and RTA as indicated (C). Extracellular viral DNA was measured by qPCR using primers specific to BALF5 (D). The value of vector control at 0 hr was set as 1. Results from three biological replicates are presented. Error bars indicate ±SD. **, p

    Techniques Used: Activation Assay, Activity Assay, Derivative Assay, Cross-linking Immunoprecipitation, Expressing, Construct, Western Blot, Inhibition, Real-time Polymerase Chain Reaction, Plasmid Preparation

    Caspases cleave m 6 A RNA modification pathway proteins (A) Diagram summarizing the major writers, readers and erasers involved in the m 6 A RNA modification pathway. (B) The downregulation of m 6 A RNA modification pathway proteins during EBV reactivation. Akata (EBV+) cells was treated with anti-IgG antibody to induce EBV reactivation for 0, 24 and 48 hrs. Western Blot was performed using antibodies as indicated. N6AMT1 and β-actin blots were included as controls. (C) Caspase inhibition blocks the degradation of m 6 A RNA modification pathway proteins. The Akata (EBV+) cells were either untreated or pretreated with a caspase-3/-7 inhibitor (Z-DEVD-FMK, 50 μM) or pan-caspase inhibitor (Z-VAD-FMK, 50 μM) for 1 hr, and then anti-IgG antibody was added for 48 hrs. Western Blot was performed using antibodies as indicated. (D and E) V5-METTL14 (D) and V5-WTAP (E) were incubated with individual caspase for 2 hrs at 37°C. Western Blot was performed using anti-METTL14, anti-V5 and anti-WTAP antibodies as indicated. The locations of antibody recognition epitopes were labelled as indicated. Arrowheads denote cleaved fragments. Star denotes non-specific bands. See also Figures S5 - S7 .
    Figure Legend Snippet: Caspases cleave m 6 A RNA modification pathway proteins (A) Diagram summarizing the major writers, readers and erasers involved in the m 6 A RNA modification pathway. (B) The downregulation of m 6 A RNA modification pathway proteins during EBV reactivation. Akata (EBV+) cells was treated with anti-IgG antibody to induce EBV reactivation for 0, 24 and 48 hrs. Western Blot was performed using antibodies as indicated. N6AMT1 and β-actin blots were included as controls. (C) Caspase inhibition blocks the degradation of m 6 A RNA modification pathway proteins. The Akata (EBV+) cells were either untreated or pretreated with a caspase-3/-7 inhibitor (Z-DEVD-FMK, 50 μM) or pan-caspase inhibitor (Z-VAD-FMK, 50 μM) for 1 hr, and then anti-IgG antibody was added for 48 hrs. Western Blot was performed using antibodies as indicated. (D and E) V5-METTL14 (D) and V5-WTAP (E) were incubated with individual caspase for 2 hrs at 37°C. Western Blot was performed using anti-METTL14, anti-V5 and anti-WTAP antibodies as indicated. The locations of antibody recognition epitopes were labelled as indicated. Arrowheads denote cleaved fragments. Star denotes non-specific bands. See also Figures S5 - S7 .

    Techniques Used: Modification, Western Blot, Inhibition, Incubation

    4) Product Images from "Caspases switch off m6A RNA modification pathway to reactivate a ubiquitous human tumor virus"

    Article Title: Caspases switch off m6A RNA modification pathway to reactivate a ubiquitous human tumor virus

    Journal: bioRxiv

    doi: 10.1101/2020.11.12.377127

    See also Figures 6 , 7 and Table S2. (A) V5-METTL3 was incubated with individual caspase for 2 hrs at 37°C. Western Blot was performed using anti-METTL3 and anti-V5 antibodies as indicated. The locations of antibody recognition epitopes were labelled as indicated. The positions of weakly cleaved fragments were labelled by arrowhead. Star denotes non-specific bands. (B) V5-tagged WTAP D301A/D302A and D301A mutants were incubated with individual recombinant caspase for 2 hrs. Western Blot was performed using antibodies as indicated. Arrowheads denote cleaved fragments. (C) Sequence alignment of WTAP sequences from 10 representative species using the Constraint-based Multiple Alignment Tool (COBALT). The cleavage motifs were highlighted by yellow color. (D) Motif analysis showing the conservation of the WTAP D302 and the surrounding amino acids. Amino acid sequences were extracted from 97 vertebrate species and motif logos were generated using WebLogo. (E-F) Akata (EBV+) cells were used to establish stable cell lines using 2 different guide RNA constructs targeting YTHDF1 (D) and ALKBH5 (E) and a non-targeting control (sg-NC). The cells were untreated or lytically induced with anti-IgG-mediated BCR activation. Cellular and viral protein expression levels were monitored by Western Blot using antibodies as indicated.
    Figure Legend Snippet: See also Figures 6 , 7 and Table S2. (A) V5-METTL3 was incubated with individual caspase for 2 hrs at 37°C. Western Blot was performed using anti-METTL3 and anti-V5 antibodies as indicated. The locations of antibody recognition epitopes were labelled as indicated. The positions of weakly cleaved fragments were labelled by arrowhead. Star denotes non-specific bands. (B) V5-tagged WTAP D301A/D302A and D301A mutants were incubated with individual recombinant caspase for 2 hrs. Western Blot was performed using antibodies as indicated. Arrowheads denote cleaved fragments. (C) Sequence alignment of WTAP sequences from 10 representative species using the Constraint-based Multiple Alignment Tool (COBALT). The cleavage motifs were highlighted by yellow color. (D) Motif analysis showing the conservation of the WTAP D302 and the surrounding amino acids. Amino acid sequences were extracted from 97 vertebrate species and motif logos were generated using WebLogo. (E-F) Akata (EBV+) cells were used to establish stable cell lines using 2 different guide RNA constructs targeting YTHDF1 (D) and ALKBH5 (E) and a non-targeting control (sg-NC). The cells were untreated or lytically induced with anti-IgG-mediated BCR activation. Cellular and viral protein expression levels were monitored by Western Blot using antibodies as indicated.

    Techniques Used: Incubation, Western Blot, Recombinant, Sequencing, Generated, Stable Transfection, Construct, Activation Assay, Expressing

    YTHDF2 restricts EBV reactivation. (A) Schematic representation showing the relative positions of Cas9 target sites for small guide RNAs sg-1 to sg-3. (B) Akata (EBV+) cells were used to establish stable cell lines using 3 different sgRNA constructs and a non-targeting control (sg-NC). The cells were untreated or lytically induced with anti-IgG-mediated cross-linking of BCR. YTHDF2 and viral protein expression levels were monitored by Western Blot using antibodies as indicated. (C) RNAs from YTHDF2-depleted and control Akata cells were extracted and analyzed by RT-qPCR. The values of control were set as 1. Error bars indicate ±SD. IE, immediate early gene; Early, early gene; Late, late gene. (D) P3HR-1 cells were used to establish stable cell lines as indicated. The cells were either untreated or treated with TPA and sodium butyrate (NaBu) to induce lytic reactivation. YTHDF2 and viral protein expression levels were monitored by Western Blot using antibodies as indicated. (E) RNAs from YTHDF2-depleted and control P3HR-1 cells were extracted and analyzed by RT-qPCR. The values of control were set as 1. Error bars indicate ±SD. IE, immediate early gene; Early, early gene; Late, late gene. (F) SUN-719 cells were used to establish stable cell lines as indicated. The cells were either untreated or treated with Gemcitabine to induce lytic reactivation. YTHDF2 and viral protein expression levels were monitored by Western Blot using antibodies as indicated. (G) Akata (EBV+) cells were used to establish control and YTHDF2 overexpression cell line as indicated. The cells were untreated or lytically induced by anti-IgG treatment. The expression of YTHDF2 as monitored by an anti-Myc antibody. Viral protein expression levels were monitored by Western Blot using antibodies as indicated. (H) Extracellular virion-associated DNA from cells treated in panel G was extracted and the relative EBV viral copy numbers were calculated by q-PCR analysis. The value of vector control at 0 hr was set as 1. Results from three biological replicates are presented. Error bars indicate ±SD. ***, p
    Figure Legend Snippet: YTHDF2 restricts EBV reactivation. (A) Schematic representation showing the relative positions of Cas9 target sites for small guide RNAs sg-1 to sg-3. (B) Akata (EBV+) cells were used to establish stable cell lines using 3 different sgRNA constructs and a non-targeting control (sg-NC). The cells were untreated or lytically induced with anti-IgG-mediated cross-linking of BCR. YTHDF2 and viral protein expression levels were monitored by Western Blot using antibodies as indicated. (C) RNAs from YTHDF2-depleted and control Akata cells were extracted and analyzed by RT-qPCR. The values of control were set as 1. Error bars indicate ±SD. IE, immediate early gene; Early, early gene; Late, late gene. (D) P3HR-1 cells were used to establish stable cell lines as indicated. The cells were either untreated or treated with TPA and sodium butyrate (NaBu) to induce lytic reactivation. YTHDF2 and viral protein expression levels were monitored by Western Blot using antibodies as indicated. (E) RNAs from YTHDF2-depleted and control P3HR-1 cells were extracted and analyzed by RT-qPCR. The values of control were set as 1. Error bars indicate ±SD. IE, immediate early gene; Early, early gene; Late, late gene. (F) SUN-719 cells were used to establish stable cell lines as indicated. The cells were either untreated or treated with Gemcitabine to induce lytic reactivation. YTHDF2 and viral protein expression levels were monitored by Western Blot using antibodies as indicated. (G) Akata (EBV+) cells were used to establish control and YTHDF2 overexpression cell line as indicated. The cells were untreated or lytically induced by anti-IgG treatment. The expression of YTHDF2 as monitored by an anti-Myc antibody. Viral protein expression levels were monitored by Western Blot using antibodies as indicated. (H) Extracellular virion-associated DNA from cells treated in panel G was extracted and the relative EBV viral copy numbers were calculated by q-PCR analysis. The value of vector control at 0 hr was set as 1. Results from three biological replicates are presented. Error bars indicate ±SD. ***, p

    Techniques Used: Stable Transfection, Construct, Expressing, Western Blot, Quantitative RT-PCR, Over Expression, Polymerase Chain Reaction, Plasmid Preparation

    Depletion of m 6 A writers and reader YTHDF3 promotes EBV reactivation. (A-E) Akata (EBV+) cells were used to establish stable cell lines using 2-3 different guide RNA constructs targeting METTL3 (A), METTL14 (B), WTAP (C), VIRMA (D) and YTHDF3 (E) and a non-targeting control (sg-NC). The cells were untreated or lytically induced with anti-IgG-mediated BCR activation. Cellular and viral protein expression levels were monitored by Western Blot using antibodies as indicated. (F) Model summarizing the regulation of m 6 A RNA modification pathway in EBV life cycle. During latency, m 6 A writers deposit the methyl group onto key viral and cellular mRNAs which are subsequently destabilized by m 6 A readers. Upon reactivation, on one hand, cellular caspases cleave the writers to limit the m 6 A modification process, and on the other hand, caspases cleave readers to limits RNA decay by CNOT-CCR4 complex, which together drive the production of massive amounts of viruses. See also Figure S7 .
    Figure Legend Snippet: Depletion of m 6 A writers and reader YTHDF3 promotes EBV reactivation. (A-E) Akata (EBV+) cells were used to establish stable cell lines using 2-3 different guide RNA constructs targeting METTL3 (A), METTL14 (B), WTAP (C), VIRMA (D) and YTHDF3 (E) and a non-targeting control (sg-NC). The cells were untreated or lytically induced with anti-IgG-mediated BCR activation. Cellular and viral protein expression levels were monitored by Western Blot using antibodies as indicated. (F) Model summarizing the regulation of m 6 A RNA modification pathway in EBV life cycle. During latency, m 6 A writers deposit the methyl group onto key viral and cellular mRNAs which are subsequently destabilized by m 6 A readers. Upon reactivation, on one hand, cellular caspases cleave the writers to limit the m 6 A modification process, and on the other hand, caspases cleave readers to limits RNA decay by CNOT-CCR4 complex, which together drive the production of massive amounts of viruses. See also Figure S7 .

    Techniques Used: Stable Transfection, Construct, Activation Assay, Expressing, Western Blot, Modification

    YTHDF2 binds to EBV transcripts and viral RNAs contain m 6 A modifications. See also Figure 5 . Akata (EBV+) cells were lytically induced by IgG-cross linking for 24 hrs. (A) Total RNA was subjected to m 6 A RIP, followed by RT-qPCR using indicated primers. Values are displayed as fold change over 10% input. GAPDH and Dicer are cellular negative and positive controls, respectively. (B) Cell lysate was collected to detect YTHDF2 binding of viral RNAs by RIP-qPCR. Values are displayed as fold change over 10% input. MALAT1 and SON are cellular negative and positive controls, respectively. Results from three biological replicates are presented. Error bars indicate ±SD. **, p
    Figure Legend Snippet: YTHDF2 binds to EBV transcripts and viral RNAs contain m 6 A modifications. See also Figure 5 . Akata (EBV+) cells were lytically induced by IgG-cross linking for 24 hrs. (A) Total RNA was subjected to m 6 A RIP, followed by RT-qPCR using indicated primers. Values are displayed as fold change over 10% input. GAPDH and Dicer are cellular negative and positive controls, respectively. (B) Cell lysate was collected to detect YTHDF2 binding of viral RNAs by RIP-qPCR. Values are displayed as fold change over 10% input. MALAT1 and SON are cellular negative and positive controls, respectively. Results from three biological replicates are presented. Error bars indicate ±SD. **, p

    Techniques Used: Quantitative RT-PCR, Binding Assay, Real-time Polymerase Chain Reaction

    YTHDF2 is cleaved by caspases in vivo and in vitro . (A) Western Blot showing YTHDF2 downregulation by IgG cross-linking induced BCR activation. Akata (EBV+) and Akata-4E3 (EBV-) cells were treated with anti-IgG antibody as indicated. YTHDF2 and viral protein expression levels were monitored by Western Blot. Arrowheads denote cleaved YTHDF2 in the longer exposure blot. (B) Caspase inhibition blocks YTHDF2 degradation. The cells were either untreated or pretreated with a pan-caspase inhibitor (Z-VAD-FMK, 50 μM) for 1 hr, and then anti-IgG antibody was added for 48 hrs. Arrowheads denote cleaved YTHDF2. (C) Functional domains and putative cleavage sites in YTHDF2. CaspDB was used to predict the potential cleavage sites in YTHDF2. The locations of the putative cleavage sites D166 and D367 were labeled as indicated. CNOT1 binding domain: responsible for the degradation of associated RNA; P/Q/N rich region: aggregation-prone region; YTH domain: responsible for binding to m 6 A-modified RNA. (D) Schematic representation of V5-tagged YTHDF2 with two putative cleavage sites. Red oval, anti-YTHDF2 monoclonal antibody recognition site. (E-F). Wild-type V5-YTHDF2 was incubated with individual recombinant caspase for 2 hrs. Western Blot was performed using either anti-YTHDF2 (E) or anti-V5 (F) antibodies. The relative position of predicted cleavage fragments was labeled as indicated. (G-H) YTHDF2 (D166A/D367A) mutant protein was incubated with individual recombinant caspase for 2 hrs. Western Blot was performed using antibodies as indicated. (I) Motif analysis showing the conservation of the two cleavage sites and the surrounding amino acids. Amino acid sequences were extracted from 97 (D166) and 80 (D367) vertebrate species and motif logos were generated using WebLogo. (J) Structure modeling of full-length YTHDF2 by I-TASSER. The two cleavage sites D166 and D367 are labeled as indicated. N and C denote N-terminus and C-terminus, respectively. (K) Triple depletion of caspase-3, −8 and −6 reduces YTHDF2 and PIAS1 degradation and blocks viral protein accumulation. The CASP3/CASP8/CASP6-triply-depleted Akata (EBV+) cells were lytically induced by anti-IgG treatment. The expression of cleaved caspases, YTHDF2, PIAS1 and viral proteins was monitored by Western Blot using antibodies as indicated. See also Figure S2 and Table S2.
    Figure Legend Snippet: YTHDF2 is cleaved by caspases in vivo and in vitro . (A) Western Blot showing YTHDF2 downregulation by IgG cross-linking induced BCR activation. Akata (EBV+) and Akata-4E3 (EBV-) cells were treated with anti-IgG antibody as indicated. YTHDF2 and viral protein expression levels were monitored by Western Blot. Arrowheads denote cleaved YTHDF2 in the longer exposure blot. (B) Caspase inhibition blocks YTHDF2 degradation. The cells were either untreated or pretreated with a pan-caspase inhibitor (Z-VAD-FMK, 50 μM) for 1 hr, and then anti-IgG antibody was added for 48 hrs. Arrowheads denote cleaved YTHDF2. (C) Functional domains and putative cleavage sites in YTHDF2. CaspDB was used to predict the potential cleavage sites in YTHDF2. The locations of the putative cleavage sites D166 and D367 were labeled as indicated. CNOT1 binding domain: responsible for the degradation of associated RNA; P/Q/N rich region: aggregation-prone region; YTH domain: responsible for binding to m 6 A-modified RNA. (D) Schematic representation of V5-tagged YTHDF2 with two putative cleavage sites. Red oval, anti-YTHDF2 monoclonal antibody recognition site. (E-F). Wild-type V5-YTHDF2 was incubated with individual recombinant caspase for 2 hrs. Western Blot was performed using either anti-YTHDF2 (E) or anti-V5 (F) antibodies. The relative position of predicted cleavage fragments was labeled as indicated. (G-H) YTHDF2 (D166A/D367A) mutant protein was incubated with individual recombinant caspase for 2 hrs. Western Blot was performed using antibodies as indicated. (I) Motif analysis showing the conservation of the two cleavage sites and the surrounding amino acids. Amino acid sequences were extracted from 97 (D166) and 80 (D367) vertebrate species and motif logos were generated using WebLogo. (J) Structure modeling of full-length YTHDF2 by I-TASSER. The two cleavage sites D166 and D367 are labeled as indicated. N and C denote N-terminus and C-terminus, respectively. (K) Triple depletion of caspase-3, −8 and −6 reduces YTHDF2 and PIAS1 degradation and blocks viral protein accumulation. The CASP3/CASP8/CASP6-triply-depleted Akata (EBV+) cells were lytically induced by anti-IgG treatment. The expression of cleaved caspases, YTHDF2, PIAS1 and viral proteins was monitored by Western Blot using antibodies as indicated. See also Figure S2 and Table S2.

    Techniques Used: In Vivo, In Vitro, Western Blot, Activation Assay, Expressing, Inhibition, Functional Assay, Labeling, Binding Assay, Modification, Incubation, Recombinant, Mutagenesis, Generated

    YTHDF2 cleavage promotes EBV replication. (A) The design of CRISPR/Cas9-resistant YTHDF2 variant was based on the sg-2 protospacer adjacent motif (PAM). D166A/D367A mutations were introduced into the PAM-mutated YTHDF2. Both constructs were cloned into a lentiviral vector with a C-terminal Myc-tag. (B-C) WT and cleavage-resistant YTHDF2 suppresses EBV replication. Akata (EBV+) YTHDF2-sg2 cells were reconstituted with WT or cleavage-resistant YTHDF2 (D166A/D367A) using lentiviral constructs. Western Blot analysis showing YTHDF2 and EBV protein expression levels in these cell lines upon IgG cross-linking as indicated (B). Extracellular and intracellular viral DNA was measured by qPCR using primers specific to BALF5 (C). The value of vector control at 0 hr was set as 1. Results from three biological replicates are presented. Error bars indicate ±SD. **, p
    Figure Legend Snippet: YTHDF2 cleavage promotes EBV replication. (A) The design of CRISPR/Cas9-resistant YTHDF2 variant was based on the sg-2 protospacer adjacent motif (PAM). D166A/D367A mutations were introduced into the PAM-mutated YTHDF2. Both constructs were cloned into a lentiviral vector with a C-terminal Myc-tag. (B-C) WT and cleavage-resistant YTHDF2 suppresses EBV replication. Akata (EBV+) YTHDF2-sg2 cells were reconstituted with WT or cleavage-resistant YTHDF2 (D166A/D367A) using lentiviral constructs. Western Blot analysis showing YTHDF2 and EBV protein expression levels in these cell lines upon IgG cross-linking as indicated (B). Extracellular and intracellular viral DNA was measured by qPCR using primers specific to BALF5 (C). The value of vector control at 0 hr was set as 1. Results from three biological replicates are presented. Error bars indicate ±SD. **, p

    Techniques Used: CRISPR, Variant Assay, Construct, Clone Assay, Plasmid Preparation, Western Blot, Expressing, Real-time Polymerase Chain Reaction

    Identification of new potential caspase substrates during EBV reactivation. (A) The cleavage motifs derived from PIAS1 (LTYD*G and NGVD*G) were used to virtually screen the entire human proteome for proteins sharing the same sequences. The human proteome dataset containing approximately 20,000 human protein-coding genes represented by the canonical protein sequence was downloaded from UniProtKB/Swiss-Prot. (B) 16 additional proteins were extracted from the screen. 8 proteins carry the LTYD*G motif (left) and 8 proteins carry the NGVD*G motif (right). 6 proteins (underlined) were selected for further validation. (C) Protein downregulation during EBV reactivation. Akata (EBV+) cells was treated with anti-IgG antibody to induce EBV reactivation for 0, 24 and 48 hrs. Western Blot showing the downregulation of 6 selected proteins using antibodies as indicated. SAMHD1 and β-actin were included as controls. Arrowhead denotes the cleaved fragment for EHMT2. (D) Caspase inhibition blocks the degradation of YTHDF2, MAGEA10, SORT1 MTA1 and EHMT2. The Akata (EBV+) cells were either untreated or pretreated with a caspase-3/-7 inhibitor (Z-DEVD-FMK, 50 μM) or pan-caspase inhibitor (Z-VAD-FMK, 50 μM) for 1 hr, and then anti-IgG antibody was added for 48 hrs. Western Blot showing the protein levels of 6 selected proteins using antibodies as indicated. SAMHD1 and β-actin were included as controls. Arrowhead denotes cleaved EHMT2 fragment.
    Figure Legend Snippet: Identification of new potential caspase substrates during EBV reactivation. (A) The cleavage motifs derived from PIAS1 (LTYD*G and NGVD*G) were used to virtually screen the entire human proteome for proteins sharing the same sequences. The human proteome dataset containing approximately 20,000 human protein-coding genes represented by the canonical protein sequence was downloaded from UniProtKB/Swiss-Prot. (B) 16 additional proteins were extracted from the screen. 8 proteins carry the LTYD*G motif (left) and 8 proteins carry the NGVD*G motif (right). 6 proteins (underlined) were selected for further validation. (C) Protein downregulation during EBV reactivation. Akata (EBV+) cells was treated with anti-IgG antibody to induce EBV reactivation for 0, 24 and 48 hrs. Western Blot showing the downregulation of 6 selected proteins using antibodies as indicated. SAMHD1 and β-actin were included as controls. Arrowhead denotes the cleaved fragment for EHMT2. (D) Caspase inhibition blocks the degradation of YTHDF2, MAGEA10, SORT1 MTA1 and EHMT2. The Akata (EBV+) cells were either untreated or pretreated with a caspase-3/-7 inhibitor (Z-DEVD-FMK, 50 μM) or pan-caspase inhibitor (Z-VAD-FMK, 50 μM) for 1 hr, and then anti-IgG antibody was added for 48 hrs. Western Blot showing the protein levels of 6 selected proteins using antibodies as indicated. SAMHD1 and β-actin were included as controls. Arrowhead denotes cleaved EHMT2 fragment.

    Techniques Used: Derivative Assay, Sequencing, Western Blot, Inhibition

    The role of EIF4H, MAGEA10, SORT1, EHMT2, and MTA1 during EBV lytic induction. See also Figure 2 . (A-E) Akata (EBV+) cells were used to establish stable cell lines using 2 or 3 different sgRNA constructs and a non-targeting control (sg-NC). The cells were untreated or lytically induced with anti-IgG treatment for 24 or 48 hrs as indicated. Cellular and viral protein expression levels were monitored by Western Blot using antibodies as indicated. (A) EIF4H depletion promotes the expression of EBV ZTA and RTA. (B) MAGEA10 depletion does not affect EBV protein expression. (C) SORT1 depletion does not significantly affect EBV protein expression. (D) EHMT2 depletion does not affect EBV protein expression. (E) MTA1 depletion does not uniformly affect EBV protein expression but slightly enhances the expression of its homolog MTA2.
    Figure Legend Snippet: The role of EIF4H, MAGEA10, SORT1, EHMT2, and MTA1 during EBV lytic induction. See also Figure 2 . (A-E) Akata (EBV+) cells were used to establish stable cell lines using 2 or 3 different sgRNA constructs and a non-targeting control (sg-NC). The cells were untreated or lytically induced with anti-IgG treatment for 24 or 48 hrs as indicated. Cellular and viral protein expression levels were monitored by Western Blot using antibodies as indicated. (A) EIF4H depletion promotes the expression of EBV ZTA and RTA. (B) MAGEA10 depletion does not affect EBV protein expression. (C) SORT1 depletion does not significantly affect EBV protein expression. (D) EHMT2 depletion does not affect EBV protein expression. (E) MTA1 depletion does not uniformly affect EBV protein expression but slightly enhances the expression of its homolog MTA2.

    Techniques Used: Stable Transfection, Construct, Expressing, Western Blot

    YTHDF2 regulates CASP8 mRNA stability through m 6 A modifications. (A-B) YTHDF2 depletion promotes CASP8 mRNA expression. Akata (EBV+) cells and P3HR-1 cells carrying different sgRNA targeting YTHDF2 or control (sg-NC) were used to extract total RNA and qPCR analyses were performed a group of YTHDF2-targeted cellular genes involved in caspase activation. The values were normalized with a non YTHDF2 target HPRT1 . The values of sg-NC were set as 1. (C-D) CASP8 is modified by m 6 A and YTHDF2 binding to CASP8 . Akata (EBV+) cells were used to perform m6A RIP-qPCR (C) and YTHDF2 RIP-qPCR (D), respectively. Values are displayed as fold change over 10% input. (E-G) YTHDF2 depletion promotes caspase-8 protein expression and PIAS1 cleavage upon lytic i nduction. Akata (EBV+) cells (E), P3HR-1 cells (F) and SNU-719 cells (G) carrying different sgRNA targeting YTHDF2 or control (sg-NC) were lytically induced by anti-IgG, TPA and sodium butyrate (NaBu) and gemcitabine treatment for 24 hrs. Protein expression was monitored by Western Blot using antibodies as indicated. (H) CASP8 m 6 A peaks were extracted from MeT-DB V2.0 database. YTHDF2-PAR-CLIP data were retrieved from Wang et al.( Wang et al., 2014 ). The Exon-7 of CASP8 with highest m 6 A peaks were analyzed for conservation among sequences derived from 100 vertebrate species. 15 potential m 6 A motifs (M1-M15) were extracted based on m 6 A motif DRACH. (I) Motif logos were generated for 15 individual sites. Red cycles denote highly conserved motifs (M2, M3, M5, M8 and M12) across 100 vertebrate species. (J-K) WT and mutant CASP8 -Exon-7 were cloned into the m 6 A-null Renilla luciferase (RLuc) reporter (3’UTR region) that also express Firefly luciferase (FLuc) from a separate promoter (J). These three reporter plasmids were transfected into parental or YTHDF2-depleted (YTHDF2 KD) SNU719 cells. Relative Renilla to Filefly luciferase activity (RLuc/FLuc) was calculated (K). The value of WT in parental cells was set as 1. (L) Model illustrating YTHDF2 regulation of CASP8 mRNA and caspase-8 regulation of YTHDF2 and PIAS1 in EBV reactivation. Results from three biological replicates are presented. Error bars indicate ±SD. *, p
    Figure Legend Snippet: YTHDF2 regulates CASP8 mRNA stability through m 6 A modifications. (A-B) YTHDF2 depletion promotes CASP8 mRNA expression. Akata (EBV+) cells and P3HR-1 cells carrying different sgRNA targeting YTHDF2 or control (sg-NC) were used to extract total RNA and qPCR analyses were performed a group of YTHDF2-targeted cellular genes involved in caspase activation. The values were normalized with a non YTHDF2 target HPRT1 . The values of sg-NC were set as 1. (C-D) CASP8 is modified by m 6 A and YTHDF2 binding to CASP8 . Akata (EBV+) cells were used to perform m6A RIP-qPCR (C) and YTHDF2 RIP-qPCR (D), respectively. Values are displayed as fold change over 10% input. (E-G) YTHDF2 depletion promotes caspase-8 protein expression and PIAS1 cleavage upon lytic i nduction. Akata (EBV+) cells (E), P3HR-1 cells (F) and SNU-719 cells (G) carrying different sgRNA targeting YTHDF2 or control (sg-NC) were lytically induced by anti-IgG, TPA and sodium butyrate (NaBu) and gemcitabine treatment for 24 hrs. Protein expression was monitored by Western Blot using antibodies as indicated. (H) CASP8 m 6 A peaks were extracted from MeT-DB V2.0 database. YTHDF2-PAR-CLIP data were retrieved from Wang et al.( Wang et al., 2014 ). The Exon-7 of CASP8 with highest m 6 A peaks were analyzed for conservation among sequences derived from 100 vertebrate species. 15 potential m 6 A motifs (M1-M15) were extracted based on m 6 A motif DRACH. (I) Motif logos were generated for 15 individual sites. Red cycles denote highly conserved motifs (M2, M3, M5, M8 and M12) across 100 vertebrate species. (J-K) WT and mutant CASP8 -Exon-7 were cloned into the m 6 A-null Renilla luciferase (RLuc) reporter (3’UTR region) that also express Firefly luciferase (FLuc) from a separate promoter (J). These three reporter plasmids were transfected into parental or YTHDF2-depleted (YTHDF2 KD) SNU719 cells. Relative Renilla to Filefly luciferase activity (RLuc/FLuc) was calculated (K). The value of WT in parental cells was set as 1. (L) Model illustrating YTHDF2 regulation of CASP8 mRNA and caspase-8 regulation of YTHDF2 and PIAS1 in EBV reactivation. Results from three biological replicates are presented. Error bars indicate ±SD. *, p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Activation Assay, Modification, Binding Assay, Western Blot, Cross-linking Immunoprecipitation, Derivative Assay, Generated, Mutagenesis, Clone Assay, Luciferase, Transfection, Activity Assay

    See also Figures 4 and 5 . (A) A group of genes in the category of “ activation of cysteine-type endopeptidase activity involved in apoptotic process ” (also called “ caspase activation ”) were extracted from YTHDF2 target genes derived from YTHDF2 RIP-seq and PAR-CLIP-seq datasets ( Liu et al., 2015 ; Liu et al., 2018 ; Wang et al., 2014 ) (B) YTHDF2 reconstitution suppresses caspase-8 expression and subsequent caspase activation. Akata (EBV+) YTHDF2-sg2 cells were reconstituted with WT or cleavage-resistant YTHDF2 (D166A/D367A) using lentiviral constructs. Western Blot analysis showing the levels for caspase-8 (CASP8), cleaved caspase-8, caspase-3 (CASP3) and cleaved caspase substrates (CASP sub.) in these cell lines upon IgG cross-linking as indicated. (C-D) Caspase-8 inhibition suppress EBV replication in YTHDF2-depleted cells. Control and YTHDF2-depleted Akata (EBV+) cells were either untreated or pretreated with caspase-8 inhibitor (Z-IETD-FMK, 50 μM) for 1 hr and then anti-IgG antibody was added for 0 to 48 hrs. Western Blot showing the protein levels of EBV ZTA and RTA as indicated (C). Extracellular viral DNA was measured by qPCR using primers specific to BALF5 (D). The value of vector control at 0 hr was set as 1. Results from three biological replicates are presented. Error bars indicate ±SD. **, p
    Figure Legend Snippet: See also Figures 4 and 5 . (A) A group of genes in the category of “ activation of cysteine-type endopeptidase activity involved in apoptotic process ” (also called “ caspase activation ”) were extracted from YTHDF2 target genes derived from YTHDF2 RIP-seq and PAR-CLIP-seq datasets ( Liu et al., 2015 ; Liu et al., 2018 ; Wang et al., 2014 ) (B) YTHDF2 reconstitution suppresses caspase-8 expression and subsequent caspase activation. Akata (EBV+) YTHDF2-sg2 cells were reconstituted with WT or cleavage-resistant YTHDF2 (D166A/D367A) using lentiviral constructs. Western Blot analysis showing the levels for caspase-8 (CASP8), cleaved caspase-8, caspase-3 (CASP3) and cleaved caspase substrates (CASP sub.) in these cell lines upon IgG cross-linking as indicated. (C-D) Caspase-8 inhibition suppress EBV replication in YTHDF2-depleted cells. Control and YTHDF2-depleted Akata (EBV+) cells were either untreated or pretreated with caspase-8 inhibitor (Z-IETD-FMK, 50 μM) for 1 hr and then anti-IgG antibody was added for 0 to 48 hrs. Western Blot showing the protein levels of EBV ZTA and RTA as indicated (C). Extracellular viral DNA was measured by qPCR using primers specific to BALF5 (D). The value of vector control at 0 hr was set as 1. Results from three biological replicates are presented. Error bars indicate ±SD. **, p

    Techniques Used: Activation Assay, Activity Assay, Derivative Assay, Cross-linking Immunoprecipitation, Expressing, Construct, Western Blot, Inhibition, Real-time Polymerase Chain Reaction, Plasmid Preparation

    Caspases cleave m 6 A RNA modification pathway proteins (A) Diagram summarizing the major writers, readers and erasers involved in the m 6 A RNA modification pathway. (B) The downregulation of m 6 A RNA modification pathway proteins during EBV reactivation. Akata (EBV+) cells was treated with anti-IgG antibody to induce EBV reactivation for 0, 24 and 48 hrs. Western Blot was performed using antibodies as indicated. N6AMT1 and β-actin blots were included as controls. (C) Caspase inhibition blocks the degradation of m 6 A RNA modification pathway proteins. The Akata (EBV+) cells were either untreated or pretreated with a caspase-3/-7 inhibitor (Z-DEVD-FMK, 50 μM) or pan-caspase inhibitor (Z-VAD-FMK, 50 μM) for 1 hr, and then anti-IgG antibody was added for 48 hrs. Western Blot was performed using antibodies as indicated. (D and E) V5-METTL14 (D) and V5-WTAP (E) were incubated with individual caspase for 2 hrs at 37°C. Western Blot was performed using anti-METTL14, anti-V5 and anti-WTAP antibodies as indicated. The locations of antibody recognition epitopes were labelled as indicated. Arrowheads denote cleaved fragments. Star denotes non-specific bands. See also Figures S5 - S7 .
    Figure Legend Snippet: Caspases cleave m 6 A RNA modification pathway proteins (A) Diagram summarizing the major writers, readers and erasers involved in the m 6 A RNA modification pathway. (B) The downregulation of m 6 A RNA modification pathway proteins during EBV reactivation. Akata (EBV+) cells was treated with anti-IgG antibody to induce EBV reactivation for 0, 24 and 48 hrs. Western Blot was performed using antibodies as indicated. N6AMT1 and β-actin blots were included as controls. (C) Caspase inhibition blocks the degradation of m 6 A RNA modification pathway proteins. The Akata (EBV+) cells were either untreated or pretreated with a caspase-3/-7 inhibitor (Z-DEVD-FMK, 50 μM) or pan-caspase inhibitor (Z-VAD-FMK, 50 μM) for 1 hr, and then anti-IgG antibody was added for 48 hrs. Western Blot was performed using antibodies as indicated. (D and E) V5-METTL14 (D) and V5-WTAP (E) were incubated with individual caspase for 2 hrs at 37°C. Western Blot was performed using anti-METTL14, anti-V5 and anti-WTAP antibodies as indicated. The locations of antibody recognition epitopes were labelled as indicated. Arrowheads denote cleaved fragments. Star denotes non-specific bands. See also Figures S5 - S7 .

    Techniques Used: Modification, Western Blot, Inhibition, Incubation

    5) Product Images from "Phosphoproteomic Profiling Reveals Epstein-Barr Virus Protein Kinase Integration of DNA Damage Response and Mitotic Signaling"

    Article Title: Phosphoproteomic Profiling Reveals Epstein-Barr Virus Protein Kinase Integration of DNA Damage Response and Mitotic Signaling

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005346

    EBV lytic reactivation triggers the phosphorylation of PP1α and a mitosis-related protein. (A) EBV reactivation triggers the phosphorylation of PP1α. Western blot analysis of cell extracts from Akata-BX1 (EBV+) and Akata-4E3 (EBV-) cells using anti-phospho-PP1α T320, anti-PP1α, anti-BGLF4 and anti-β-actin antibodies as indicated. The cells were untreated (0 hr) or treated with IgG (1:200) for 24, 48 and 72 hrs as indicated. Arrow heads indicate the positions of phospho-PP1α T320. (B) EBV reactivation and BGLF4 induction trigger the phosphorylation of a mitosis-related protein. Western blot analysis of cell extracts from Akata-BX1 (EBV+) and Akata-4E3 (EBV-) cells using anti-phospho-Ser/Thr-Pro MPM2 antibody, anti-BGLF4 and anti-β-actin antibodies as indicated. The cells were untreated (0 hr) or treated with IgG or Doxycycline as indicated. Arrow head indicates the position of a phosphorylated mitotic protein.
    Figure Legend Snippet: EBV lytic reactivation triggers the phosphorylation of PP1α and a mitosis-related protein. (A) EBV reactivation triggers the phosphorylation of PP1α. Western blot analysis of cell extracts from Akata-BX1 (EBV+) and Akata-4E3 (EBV-) cells using anti-phospho-PP1α T320, anti-PP1α, anti-BGLF4 and anti-β-actin antibodies as indicated. The cells were untreated (0 hr) or treated with IgG (1:200) for 24, 48 and 72 hrs as indicated. Arrow heads indicate the positions of phospho-PP1α T320. (B) EBV reactivation and BGLF4 induction trigger the phosphorylation of a mitosis-related protein. Western blot analysis of cell extracts from Akata-BX1 (EBV+) and Akata-4E3 (EBV-) cells using anti-phospho-Ser/Thr-Pro MPM2 antibody, anti-BGLF4 and anti-β-actin antibodies as indicated. The cells were untreated (0 hr) or treated with IgG or Doxycycline as indicated. Arrow head indicates the position of a phosphorylated mitotic protein.

    Techniques Used: Western Blot

    6) Product Images from "Characterising the KMP-11 and HSP-70 recombinant antigens' humoral immune response profile in chagasic patients"

    Article Title: Characterising the KMP-11 and HSP-70 recombinant antigens' humoral immune response profile in chagasic patients

    Journal: BMC Infectious Diseases

    doi: 10.1186/1471-2334-9-186

    IgG isotypes profile against T. cruzi KMP-11 recombinant protein . IgG1 (5A), IgG2 (5B), IgG3 (5C) and IgG4 (5D) isotype levels for patients in acute (AC), indeterminate (IND) and cardiac chronic phase (CCC) and healthy individuals (HD). Values are given as optical densities at 492 nm. The dotted line represents the cut-off values based on the mean of healthy individual values plus 3 standard deviations. Horizontal lines on each group represent the mean and standard deviation values, the mean being the larger one.
    Figure Legend Snippet: IgG isotypes profile against T. cruzi KMP-11 recombinant protein . IgG1 (5A), IgG2 (5B), IgG3 (5C) and IgG4 (5D) isotype levels for patients in acute (AC), indeterminate (IND) and cardiac chronic phase (CCC) and healthy individuals (HD). Values are given as optical densities at 492 nm. The dotted line represents the cut-off values based on the mean of healthy individual values plus 3 standard deviations. Horizontal lines on each group represent the mean and standard deviation values, the mean being the larger one.

    Techniques Used: Recombinant, Countercurrent Chromatography, Standard Deviation

    ELISA for total IgG antibody levels from acute (AC), indeterminate (IND) and cardiac chronic (CCC) chagasic patients and healthy individuals (HD) against T. cruzi lysate (2A), KMP-11 (2B), truncated T. cruzi HSP-70 (TcHSP-70T) (2C), and T. rangeli HSP-70 (2D) recombinant proteins . Values are given as optical densities at 405 nm. The dotted line represents the cut-off values based on the mean of healthy individual values plus 3 standard deviations. Horizontal lines on each group represent mean and standard deviation values, the mean being the larger one. Statistically significant differences among chagasic groups are represented by an asterisk.
    Figure Legend Snippet: ELISA for total IgG antibody levels from acute (AC), indeterminate (IND) and cardiac chronic (CCC) chagasic patients and healthy individuals (HD) against T. cruzi lysate (2A), KMP-11 (2B), truncated T. cruzi HSP-70 (TcHSP-70T) (2C), and T. rangeli HSP-70 (2D) recombinant proteins . Values are given as optical densities at 405 nm. The dotted line represents the cut-off values based on the mean of healthy individual values plus 3 standard deviations. Horizontal lines on each group represent mean and standard deviation values, the mean being the larger one. Statistically significant differences among chagasic groups are represented by an asterisk.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Countercurrent Chromatography, Recombinant, Standard Deviation

    IgG isotype profile against T. cruzi lysate . IgG1 (4A), IgG2 (4B), IgG3 (4C) and IgG4 (4D) isotype levels for patients in acute (AC), indeterminate (IND) and cardiac chronic phase (CCC) and healthy individuals (HD). Values are given as optical densities at 492 nm. The dotted line represents the cut-off values based on the mean of healthy individual values plus 3 standard deviations. Horizontal lines on each group represent mean and standard deviation values, the mean being the larger one.
    Figure Legend Snippet: IgG isotype profile against T. cruzi lysate . IgG1 (4A), IgG2 (4B), IgG3 (4C) and IgG4 (4D) isotype levels for patients in acute (AC), indeterminate (IND) and cardiac chronic phase (CCC) and healthy individuals (HD). Values are given as optical densities at 492 nm. The dotted line represents the cut-off values based on the mean of healthy individual values plus 3 standard deviations. Horizontal lines on each group represent mean and standard deviation values, the mean being the larger one.

    Techniques Used: Countercurrent Chromatography, Standard Deviation

    IgG isotypes profile against T. rangeli HSP-70 recombinant protein . IgG1 (6A), IgG2 (6B), IgG3 (6C) and IgG4 (6D) isotype levels of patients in acute (AC), indeterminate (IND) and cardiac chronic phase (CCC) and healthy individuals (HD). Values are given as optical densities at 492 nm. The dotted line represents the cut-off values based on the mean of healthy individual values plus 3 standard deviations. Horizontal lines on each group represent the mean and standard deviation values, the mean being the larger one.
    Figure Legend Snippet: IgG isotypes profile against T. rangeli HSP-70 recombinant protein . IgG1 (6A), IgG2 (6B), IgG3 (6C) and IgG4 (6D) isotype levels of patients in acute (AC), indeterminate (IND) and cardiac chronic phase (CCC) and healthy individuals (HD). Values are given as optical densities at 492 nm. The dotted line represents the cut-off values based on the mean of healthy individual values plus 3 standard deviations. Horizontal lines on each group represent the mean and standard deviation values, the mean being the larger one.

    Techniques Used: Recombinant, Countercurrent Chromatography, Standard Deviation

    7) Product Images from "Monitoring of Anti-Hepatitis E Virus Antibody Seroconversion in Asymptomatically Infected Blood Donors: Systematic Comparison of Nine Commercial Anti-HEV IgM and IgG Assays"

    Article Title: Monitoring of Anti-Hepatitis E Virus Antibody Seroconversion in Asymptomatically Infected Blood Donors: Systematic Comparison of Nine Commercial Anti-HEV IgM and IgG Assays

    Journal: Viruses

    doi: 10.3390/v8080232

    Comparison of different anti-hepatitis E virus (HEV) assays regarding the course of immune response during seroconversion of 10 blood donors with acute HEV infection. The course of immune response of 10 blood donors with autochthonous HEV infection is displayed, determined by 10 different commercially available anti-HEV immunoassays. The day of the detection of HEV RNA by PCR screening was defined as day 0 [ 28 ], confirmation of the presence of HEV RNA is indicated by gray shading. The period of positive testing results is displayed by light grey bars for the five HEV immunoglogulin (Ig)M-specific assays, by white bars for the four HEV IgG-specific assays and by dark grey bars for the HEV all antibody assay (see Table 1 for the encoding of the kits). Bars are starting at half of the interval between the last negative and first positive sample and last until half of the interval between last positive and first negative sample. The Assure HEV IgM Rapid Test (MP-Bior-IgM) was only performed with limited samples for donor 3 (day 0–126), donor 5 (day 0–40), donor 8 (day 28–52) and donor 9 (day 0–57). SC/O: signal-to-cutoff; AB: antibody.
    Figure Legend Snippet: Comparison of different anti-hepatitis E virus (HEV) assays regarding the course of immune response during seroconversion of 10 blood donors with acute HEV infection. The course of immune response of 10 blood donors with autochthonous HEV infection is displayed, determined by 10 different commercially available anti-HEV immunoassays. The day of the detection of HEV RNA by PCR screening was defined as day 0 [ 28 ], confirmation of the presence of HEV RNA is indicated by gray shading. The period of positive testing results is displayed by light grey bars for the five HEV immunoglogulin (Ig)M-specific assays, by white bars for the four HEV IgG-specific assays and by dark grey bars for the HEV all antibody assay (see Table 1 for the encoding of the kits). Bars are starting at half of the interval between the last negative and first positive sample and last until half of the interval between last positive and first negative sample. The Assure HEV IgM Rapid Test (MP-Bior-IgM) was only performed with limited samples for donor 3 (day 0–126), donor 5 (day 0–40), donor 8 (day 28–52) and donor 9 (day 0–57). SC/O: signal-to-cutoff; AB: antibody.

    Techniques Used: Infection, Polymerase Chain Reaction

    8) Product Images from "Tricomponent Immunopotentiating System as a Novel Molecular Design Strategy for Malaria Vaccine Development ▿"

    Article Title: Tricomponent Immunopotentiating System as a Novel Molecular Design Strategy for Malaria Vaccine Development ▿

    Journal: Infection and Immunity

    doi: 10.1128/IAI.05214-11

    Parasite recognition, IgG subclasses, and maintenance of the antisera induced by the COMP-Z-based tricomponent complex. The antisera obtained from the immunized mice in the experiments described in the legend to were analyzed for parasite recognition
    Figure Legend Snippet: Parasite recognition, IgG subclasses, and maintenance of the antisera induced by the COMP-Z-based tricomponent complex. The antisera obtained from the immunized mice in the experiments described in the legend to were analyzed for parasite recognition

    Techniques Used: Mouse Assay

    Immunogenicity of the tricomponent complex. Mice were immunized by the subcutaneous or intranasal route three times, at weeks 0, 2, and 4, and antisera were collected 2 weeks after the third immunization to evaluate the Pvs25-specific IgG titers. All
    Figure Legend Snippet: Immunogenicity of the tricomponent complex. Mice were immunized by the subcutaneous or intranasal route three times, at weeks 0, 2, and 4, and antisera were collected 2 weeks after the third immunization to evaluate the Pvs25-specific IgG titers. All

    Techniques Used: Mouse Assay

    9) Product Images from "Cell cycle-dependent transcription factors control the expression of yeast telomerase RNA"

    Article Title: Cell cycle-dependent transcription factors control the expression of yeast telomerase RNA

    Journal: RNA

    doi: 10.1261/rna.037663.112

    The E region containing MCB sites confers cell cycle regulation to TLC1 transcription. ( A ) Scheme of the RBP-ChIP experiment. Representation of the MS2-tagged (gray box) TLC1 locus transcribed by RNA polymerase II. Following transcription, the MS2 stems are folded, and MS2-ProA proteins are bound to the stems. Performing a chromatin immunoprecipitation (ChIP) with IgG on cells that are transcribing TLC1 will coimmunoprecipitate DNA sequences close to the 3′-end sequence of TLC1 . This immunoprecipitation is expected to be dependent on the RNA, treatment with RNase A should abolish immunoprecipitation and is used as control. The primer pair 1 (PP1) overlaps the promoter and the beginning of the gene, whereas the primer pair 2 (PP2) is just downstream from the 10xMS2 tag. ( B ) qPCR analysis of RBP-ChIP experiments on SBY40 (untagged TLC1) and SBY44 (TLC1-10xMS2) cells at different time points after release from the G1 arrest ( t = 0). The amount of coprecipitating DNA was measured by qPCR using primer pair 1 (PP1) and primer pair 2 (PP2) located at 1.1 kb and 60 nt from the MS2 tag, respectively. Each sample was also treated with RNase A to determine the dependence of the immunoprecipitation on the RNA. Average values of three independent biological replicates (two for RNase-treated) normalized against input DNA with SD are shown. ( C ) qRT-PCR analysis of Cln2 RNA levels at indicated time points for the same experiments as shown in B . CLN2 is a well-known SBF cell cycle–regulated gene and thus serves as a positive control for cell synchronization and activation of the transcription. Average values of three independent biological replicates normalized against Act1 with standard deviation are shown as fold change over t = 0 (G1). ( D ) qRT-PCR analysis of lacZ RNA levels at indicated time points. MLY30 cells carrying SLP162 ( CYC1 pro- lacZ ), SLP164 ( CYC1 UAS TLC1E pro- lacZ ), or SLP185 ( CYC1 UAS TLC1E -mcb pro-lacZ) were arrested in G1. Synchronized cultures were released, and samples were taken every 20 min. Average values of three independent biological replicates normalized against Act1 mRNA with SD are shown as fold change over t = 0 (G1). ( E ) Northern blot analysis of the same samples as in D .
    Figure Legend Snippet: The E region containing MCB sites confers cell cycle regulation to TLC1 transcription. ( A ) Scheme of the RBP-ChIP experiment. Representation of the MS2-tagged (gray box) TLC1 locus transcribed by RNA polymerase II. Following transcription, the MS2 stems are folded, and MS2-ProA proteins are bound to the stems. Performing a chromatin immunoprecipitation (ChIP) with IgG on cells that are transcribing TLC1 will coimmunoprecipitate DNA sequences close to the 3′-end sequence of TLC1 . This immunoprecipitation is expected to be dependent on the RNA, treatment with RNase A should abolish immunoprecipitation and is used as control. The primer pair 1 (PP1) overlaps the promoter and the beginning of the gene, whereas the primer pair 2 (PP2) is just downstream from the 10xMS2 tag. ( B ) qPCR analysis of RBP-ChIP experiments on SBY40 (untagged TLC1) and SBY44 (TLC1-10xMS2) cells at different time points after release from the G1 arrest ( t = 0). The amount of coprecipitating DNA was measured by qPCR using primer pair 1 (PP1) and primer pair 2 (PP2) located at 1.1 kb and 60 nt from the MS2 tag, respectively. Each sample was also treated with RNase A to determine the dependence of the immunoprecipitation on the RNA. Average values of three independent biological replicates (two for RNase-treated) normalized against input DNA with SD are shown. ( C ) qRT-PCR analysis of Cln2 RNA levels at indicated time points for the same experiments as shown in B . CLN2 is a well-known SBF cell cycle–regulated gene and thus serves as a positive control for cell synchronization and activation of the transcription. Average values of three independent biological replicates normalized against Act1 with standard deviation are shown as fold change over t = 0 (G1). ( D ) qRT-PCR analysis of lacZ RNA levels at indicated time points. MLY30 cells carrying SLP162 ( CYC1 pro- lacZ ), SLP164 ( CYC1 UAS TLC1E pro- lacZ ), or SLP185 ( CYC1 UAS TLC1E -mcb pro-lacZ) were arrested in G1. Synchronized cultures were released, and samples were taken every 20 min. Average values of three independent biological replicates normalized against Act1 mRNA with SD are shown as fold change over t = 0 (G1). ( E ) Northern blot analysis of the same samples as in D .

    Techniques Used: Chromatin Immunoprecipitation, Sequencing, Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Positive Control, Activation Assay, Standard Deviation, Northern Blot

    10) Product Images from "Differences in serum IgA responses to HIV-1 gp41 in elite controllers compared to viral suppressors on highly active antiretroviral therapy"

    Article Title: Differences in serum IgA responses to HIV-1 gp41 in elite controllers compared to viral suppressors on highly active antiretroviral therapy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0180245

    HIV proteins recognized by IgA. Western blotting was used to evaluate T1 and T2 serum IgA antibodies to HIV IIIB Env antigens (gp160, gp120 surface unit and gp41 transmembrane protein), Pol proteins (p66 and p51 reverse transcriptase subunits, p31 integrase) and Gag proteins (p55 precursor, p24 capsid and p17 matrix). (A) Representative reactivity to HIV proteins by IgA in 6 different EC and HN subjects. The 9 HIV proteins on the strips are designated on the left using an HIV IgG positive control serum. (B) The number of HIV antigens bound by IgA at both T1 and T2 was compared using the Mann-Whitney test and is depicted in a Tukey box plot. (C) The percentage of subjects positive for IgA antibodies to each HIV protein at T1 and T2 was compared using Fisher's exact test. *p
    Figure Legend Snippet: HIV proteins recognized by IgA. Western blotting was used to evaluate T1 and T2 serum IgA antibodies to HIV IIIB Env antigens (gp160, gp120 surface unit and gp41 transmembrane protein), Pol proteins (p66 and p51 reverse transcriptase subunits, p31 integrase) and Gag proteins (p55 precursor, p24 capsid and p17 matrix). (A) Representative reactivity to HIV proteins by IgA in 6 different EC and HN subjects. The 9 HIV proteins on the strips are designated on the left using an HIV IgG positive control serum. (B) The number of HIV antigens bound by IgA at both T1 and T2 was compared using the Mann-Whitney test and is depicted in a Tukey box plot. (C) The percentage of subjects positive for IgA antibodies to each HIV protein at T1 and T2 was compared using Fisher's exact test. *p

    Techniques Used: Western Blot, Positive Control, MANN-WHITNEY

    Specificity and avidity of gp41 antibodies. Tukey box plots illustrating the (A) IgG or (B) IgA sp. act. to gp41 HR1 full length peptide, gp41 ID cyclic peptide and the 54Q protein containing the MPER and HR2 regions at T2. Antibodies to 54Q were measured using ELISA; those to HR1 and ID were measured simultaneously using BAMA. The fluorescent intensity (FI) values obtained in assays for HR1- and ID-specific IgA or IgG were adjusted relative to the amount of total IgA or IgG in the diluted serum samples to obtain the sp. act. Note that the IgG responses in EC and subjects on HAART were not significantly different. (C) The avidity of anti-HR1 and anti-ID IgA antibodies was measured using the NaSCN displacement ELISA. *p
    Figure Legend Snippet: Specificity and avidity of gp41 antibodies. Tukey box plots illustrating the (A) IgG or (B) IgA sp. act. to gp41 HR1 full length peptide, gp41 ID cyclic peptide and the 54Q protein containing the MPER and HR2 regions at T2. Antibodies to 54Q were measured using ELISA; those to HR1 and ID were measured simultaneously using BAMA. The fluorescent intensity (FI) values obtained in assays for HR1- and ID-specific IgA or IgG were adjusted relative to the amount of total IgA or IgG in the diluted serum samples to obtain the sp. act. Note that the IgG responses in EC and subjects on HAART were not significantly different. (C) The avidity of anti-HR1 and anti-ID IgA antibodies was measured using the NaSCN displacement ELISA. *p

    Techniques Used: Activated Clotting Time Assay, Enzyme-linked Immunosorbent Assay

    Persistence of IgA and IgG antibody concentrations. Concentrations of serum IgG and IgA antibodies to (A) gp120 consensus B, (B) gp41 BAL (C) gp70-V1V2 Clade B/Case A2 and (D) p24 HXB2 were quantitated at T1 and T2 using ELISA. (E) Total IgG and IgA concentrations at T1 and T2 were also measured by ELISA. Dashed lines denote the cut-offs for significance, which represent the mean + 3 SD of Neg control values at both time points, or at T1 alone if T2 was not tested (nt). In every infection group, concentrations of antigen-specific IgG or IgA were significantly greater than those in Neg controls (all p
    Figure Legend Snippet: Persistence of IgA and IgG antibody concentrations. Concentrations of serum IgG and IgA antibodies to (A) gp120 consensus B, (B) gp41 BAL (C) gp70-V1V2 Clade B/Case A2 and (D) p24 HXB2 were quantitated at T1 and T2 using ELISA. (E) Total IgG and IgA concentrations at T1 and T2 were also measured by ELISA. Dashed lines denote the cut-offs for significance, which represent the mean + 3 SD of Neg control values at both time points, or at T1 alone if T2 was not tested (nt). In every infection group, concentrations of antigen-specific IgG or IgA were significantly greater than those in Neg controls (all p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Infection

    Magnitude of HIV-specific antibody responses. The average IgG and IgA sp. act. at T1 and T2 to (A) gp120 consensus B , (B) gp41 BAL , (C) gp70-V1V2 Case A2 , (D) p24 HXB2 , (E) nef and (F) gp41 ELDKWA peptide is presented as the percentage of antibody within each Ig isotype. Also shown is the sp. act. to (G) gp41 QLAVERY peptide at T1 and (H) C1 peptides at T2. Bars denote medians. Dashed lines represent the mean + 3SD of Neg controls. The frequency of subjects with positive responses is presented below each graph. *p
    Figure Legend Snippet: Magnitude of HIV-specific antibody responses. The average IgG and IgA sp. act. at T1 and T2 to (A) gp120 consensus B , (B) gp41 BAL , (C) gp70-V1V2 Case A2 , (D) p24 HXB2 , (E) nef and (F) gp41 ELDKWA peptide is presented as the percentage of antibody within each Ig isotype. Also shown is the sp. act. to (G) gp41 QLAVERY peptide at T1 and (H) C1 peptides at T2. Bars denote medians. Dashed lines represent the mean + 3SD of Neg controls. The frequency of subjects with positive responses is presented below each graph. *p

    Techniques Used: Activated Clotting Time Assay

    Avidity of HIV-specific IgG and IgA antibodies. The average avidity of serum IgG and IgA antibodies to (A) gp120 consensus B , (B) 293T-derived gp41 BAL , (C) p24, (D) E . coli -derived gp41 IIIB and (E) gp70-V1V2 Case A2 at T1 and T2 is shown for subjects that had levels of binding antibodies that produced absorbance values > 0.5 at a 1/50 (for IgA) or 1/100 (for IgG) dilution. Bars represent medians. *p
    Figure Legend Snippet: Avidity of HIV-specific IgG and IgA antibodies. The average avidity of serum IgG and IgA antibodies to (A) gp120 consensus B , (B) 293T-derived gp41 BAL , (C) p24, (D) E . coli -derived gp41 IIIB and (E) gp70-V1V2 Case A2 at T1 and T2 is shown for subjects that had levels of binding antibodies that produced absorbance values > 0.5 at a 1/50 (for IgA) or 1/100 (for IgG) dilution. Bars represent medians. *p

    Techniques Used: Derivative Assay, Binding Assay, Produced

    Antibody responses to bacterial RNA polymerase and HR1 peptides. (A) IgG and (B) IgA antibodies to E . coli RNA polymerase were measured by ELISA in T2 serum and are expressed as the % of antibodies within the total IgG or IgA. There were no significant differences between the groups. (C) IgA antibodies to HR1 overlapping peptides were measured by ELISA using a 1/50 serum dilution. Results for the QARLAVERY peptide are not presented since responses to this peptide were already shown to be absent. To determine the magnitude of peptide-specific IgA antibody responses, the absorbance measured for QARLAVERY peptide (the background control) was subtracted from the absorbance obtained for each peptide (P). The adjusted absorbance value was then divided by the concentration of total IgA in the diluted serum. No significant differences were found among the groups for responses to P1-7. HC did have greater IgA responses to P8 when compared to EC (**p
    Figure Legend Snippet: Antibody responses to bacterial RNA polymerase and HR1 peptides. (A) IgG and (B) IgA antibodies to E . coli RNA polymerase were measured by ELISA in T2 serum and are expressed as the % of antibodies within the total IgG or IgA. There were no significant differences between the groups. (C) IgA antibodies to HR1 overlapping peptides were measured by ELISA using a 1/50 serum dilution. Results for the QARLAVERY peptide are not presented since responses to this peptide were already shown to be absent. To determine the magnitude of peptide-specific IgA antibody responses, the absorbance measured for QARLAVERY peptide (the background control) was subtracted from the absorbance obtained for each peptide (P). The adjusted absorbance value was then divided by the concentration of total IgA in the diluted serum. No significant differences were found among the groups for responses to P1-7. HC did have greater IgA responses to P8 when compared to EC (**p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Concentration Assay

    HIV-specific IgG and IgA responses in individuals with normal or elevated total IgG concentration. The gp120, gp41 and p24 sp. act. of (A) IgG and (B) IgA antibodies measured in subjects with normal levels of total IgG (Nor; open circles) is shown in comparison to those with IgG hypergammaglobulinemia (↑ IgG; closed circles). The triangles in the HN graphs represent the 4 subjects with IgA hypergammaglobulinemia. Bars are medians. *p
    Figure Legend Snippet: HIV-specific IgG and IgA responses in individuals with normal or elevated total IgG concentration. The gp120, gp41 and p24 sp. act. of (A) IgG and (B) IgA antibodies measured in subjects with normal levels of total IgG (Nor; open circles) is shown in comparison to those with IgG hypergammaglobulinemia (↑ IgG; closed circles). The triangles in the HN graphs represent the 4 subjects with IgA hypergammaglobulinemia. Bars are medians. *p

    Techniques Used: Concentration Assay, Activated Clotting Time Assay

    Specificity and avidity of gp41 antibodies. Tukey box plots illustrating the (A) IgG or (B) IgA sp. act. to gp41 HR1 full length peptide, gp41 ID cyclic peptide and the 54Q protein containing the MPER and HR2 regions at T2. Antibodies to 54Q were measured using ELISA; those to HR1 and ID were measured simultaneously using BAMA. The fluorescent intensity (FI) values obtained in assays for HR1- and ID-specific IgA or IgG were adjusted relative to the amount of total IgA or IgG in the diluted serum samples to obtain the sp. act. Note that the IgG responses in EC and subjects on HAART were not significantly different. (C) The avidity of anti-HR1 and anti-ID IgA antibodies was measured using the NaSCN displacement ELISA. *p
    Figure Legend Snippet: Specificity and avidity of gp41 antibodies. Tukey box plots illustrating the (A) IgG or (B) IgA sp. act. to gp41 HR1 full length peptide, gp41 ID cyclic peptide and the 54Q protein containing the MPER and HR2 regions at T2. Antibodies to 54Q were measured using ELISA; those to HR1 and ID were measured simultaneously using BAMA. The fluorescent intensity (FI) values obtained in assays for HR1- and ID-specific IgA or IgG were adjusted relative to the amount of total IgA or IgG in the diluted serum samples to obtain the sp. act. Note that the IgG responses in EC and subjects on HAART were not significantly different. (C) The avidity of anti-HR1 and anti-ID IgA antibodies was measured using the NaSCN displacement ELISA. *p

    Techniques Used: Activated Clotting Time Assay, Enzyme-linked Immunosorbent Assay

    Persistence of IgA and IgG antibody concentrations. Concentrations of serum IgG and IgA antibodies to (A) gp120 consensus B, (B) gp41 BAL (C) gp70-V1V2 Clade B/Case A2 and (D) p24 HXB2 were quantitated at T1 and T2 using ELISA. (E) Total IgG and IgA concentrations at T1 and T2 were also measured by ELISA. Dashed lines denote the cut-offs for significance, which represent the mean + 3 SD of Neg control values at both time points, or at T1 alone if T2 was not tested (nt). In every infection group, concentrations of antigen-specific IgG or IgA were significantly greater than those in Neg controls (all p
    Figure Legend Snippet: Persistence of IgA and IgG antibody concentrations. Concentrations of serum IgG and IgA antibodies to (A) gp120 consensus B, (B) gp41 BAL (C) gp70-V1V2 Clade B/Case A2 and (D) p24 HXB2 were quantitated at T1 and T2 using ELISA. (E) Total IgG and IgA concentrations at T1 and T2 were also measured by ELISA. Dashed lines denote the cut-offs for significance, which represent the mean + 3 SD of Neg control values at both time points, or at T1 alone if T2 was not tested (nt). In every infection group, concentrations of antigen-specific IgG or IgA were significantly greater than those in Neg controls (all p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Infection

    Antibody responses to bacterial RNA polymerase and HR1 peptides. (A) IgG and (B) IgA antibodies to E . coli RNA polymerase were measured by ELISA in T2 serum and are expressed as the % of antibodies within the total IgG or IgA. There were no significant differences between the groups. (C) IgA antibodies to HR1 overlapping peptides were measured by ELISA using a 1/50 serum dilution. Results for the QARLAVERY peptide are not presented since responses to this peptide were already shown to be absent. To determine the magnitude of peptide-specific IgA antibody responses, the absorbance measured for QARLAVERY peptide (the background control) was subtracted from the absorbance obtained for each peptide (P). The adjusted absorbance value was then divided by the concentration of total IgA in the diluted serum. No significant differences were found among the groups for responses to P1-7. HC did have greater IgA responses to P8 when compared to EC (**p
    Figure Legend Snippet: Antibody responses to bacterial RNA polymerase and HR1 peptides. (A) IgG and (B) IgA antibodies to E . coli RNA polymerase were measured by ELISA in T2 serum and are expressed as the % of antibodies within the total IgG or IgA. There were no significant differences between the groups. (C) IgA antibodies to HR1 overlapping peptides were measured by ELISA using a 1/50 serum dilution. Results for the QARLAVERY peptide are not presented since responses to this peptide were already shown to be absent. To determine the magnitude of peptide-specific IgA antibody responses, the absorbance measured for QARLAVERY peptide (the background control) was subtracted from the absorbance obtained for each peptide (P). The adjusted absorbance value was then divided by the concentration of total IgA in the diluted serum. No significant differences were found among the groups for responses to P1-7. HC did have greater IgA responses to P8 when compared to EC (**p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Concentration Assay

    11) Product Images from "Double-Antigen Enzyme-Linked Immunosorbent Assay for Detection of Hepatitis E Virus-Specific Antibodies in Human or Swine Sera "

    Article Title: Double-Antigen Enzyme-Linked Immunosorbent Assay for Detection of Hepatitis E Virus-Specific Antibodies in Human or Swine Sera

    Journal: Clinical and Vaccine Immunology : CVI

    doi: 10.1128/CVI.00186-07

    Titration curves of the WHO reference reagent for HEV antibody (NIBSC code 95/584) obtained by the new ELISA and the reference HEV ELISA for IgG. Each data point is the average OD reading for triplicate tests, and the error bars represent the standard
    Figure Legend Snippet: Titration curves of the WHO reference reagent for HEV antibody (NIBSC code 95/584) obtained by the new ELISA and the reference HEV ELISA for IgG. Each data point is the average OD reading for triplicate tests, and the error bars represent the standard

    Techniques Used: Titration, Enzyme-linked Immunosorbent Assay

    Scatter chart of OD values obtained by the new sandwich ELISA for sera known to contain anti-HEV IgM or IgG antibodies, or both, and sera from other patients or healthy controls. The solid horizontal line represents the COV. Neg, negative; Pos, positive.
    Figure Legend Snippet: Scatter chart of OD values obtained by the new sandwich ELISA for sera known to contain anti-HEV IgM or IgG antibodies, or both, and sera from other patients or healthy controls. The solid horizontal line represents the COV. Neg, negative; Pos, positive.

    Techniques Used: Sandwich ELISA

    12) Product Images from "Exopolysaccharide from Lactobacillus rhamnosus KL37 Inhibits T Cell-dependent Immune Response in Mice"

    Article Title: Exopolysaccharide from Lactobacillus rhamnosus KL37 Inhibits T Cell-dependent Immune Response in Mice

    Journal: Archivum Immunologiae et Therapiae Experimentalis

    doi: 10.1007/s00005-020-00581-7

    Hypothetical model of EPS-37 modulation of immune response in the course of CIA. Purified EPS-37 inhibits T-cell proliferation and a production of IFN-γ. Such action favors M2 macrophage polarization (an anti-inflammatory effect) and facilitates suppression of arthritogenic CII-specific IgG (T cell-dependent humoral response). Suppression = hash mark, Activation = solid line
    Figure Legend Snippet: Hypothetical model of EPS-37 modulation of immune response in the course of CIA. Purified EPS-37 inhibits T-cell proliferation and a production of IFN-γ. Such action favors M2 macrophage polarization (an anti-inflammatory effect) and facilitates suppression of arthritogenic CII-specific IgG (T cell-dependent humoral response). Suppression = hash mark, Activation = solid line

    Techniques Used: Purification, Activation Assay

    Differential effect of intravenous and subcutaneous administration of EPS-37 on the production of CII-specific IgG in the course of CIA. Mice immunized with CII in the presence of CFA (day 0, first immunization, sc) and with CII in the presence of LPS (day 21, second immunization, ip) were given EPS-37 systemically, intravenously ( a ) or subcutaneously ( b ) three times a week starting on the day of second immunization (day 21) till the end of the experiment. The level of anti-CII antibodies: IgG (black bars), IgG2a (hashed bars), IgG1 (gray bars) in serum is shown as a percentage of positive control (saline injected mice; white bars). Data represent one out of three similar experiments. Results are expressed as a mean of the measurements of each individual mouse serum ± SEM. * P
    Figure Legend Snippet: Differential effect of intravenous and subcutaneous administration of EPS-37 on the production of CII-specific IgG in the course of CIA. Mice immunized with CII in the presence of CFA (day 0, first immunization, sc) and with CII in the presence of LPS (day 21, second immunization, ip) were given EPS-37 systemically, intravenously ( a ) or subcutaneously ( b ) three times a week starting on the day of second immunization (day 21) till the end of the experiment. The level of anti-CII antibodies: IgG (black bars), IgG2a (hashed bars), IgG1 (gray bars) in serum is shown as a percentage of positive control (saline injected mice; white bars). Data represent one out of three similar experiments. Results are expressed as a mean of the measurements of each individual mouse serum ± SEM. * P

    Techniques Used: Mouse Assay, Positive Control, Injection

    13) Product Images from "Inhibition of Heavy Chain and ?2-Microglobulin Synthesis as a Mechanism of Major Histocompatibility Complex Class I Downregulation during Epstein-Barr Virus Replication ▿"

    Article Title: Inhibition of Heavy Chain and ?2-Microglobulin Synthesis as a Mechanism of Major Histocompatibility Complex Class I Downregulation during Epstein-Barr Virus Replication ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01999-06

    MHC class I downregulation is independent of virus DNA replication. A. MHC class I downregulation is an early event in the virus lytic cycle. The lytic cycle was induced in Akata Bx1g cells by IgG cross-linking, and the levels of expression of immediate-early (BZLF1), early (EA-R-p85), and late (gp350/220) lytic cycle antigens were assessed by intracellular staining. In parallel, surface MHC class I was detected at each time point after induction. Percentages of cells positive for each of the antigens at the indicated time points are shown on the left y axis, whereas the relative MHC class I downregulation in GFP-positive cells compared with the GFP-negative cells is shown on the right y axis. B. MHC class I downregulation is independent of virus DNA replication and late gene expression. Akata Bx1g cells were induced by IgG cross-linking in the presence or absence of 0.2 mM acyclovir. At the indicated time points, cells were collected and stained for intracellular BZLF1 or gp350/220 expression. The numbers inside the dot plots indicate percentages of cells in the relevant quadrants. Mean fluorescence intensity of BZLF1-specific staining is shown for cells positive for both GFP and BZLF1. In parallel, samples were also stained for surface MHC class I, and the percentage of MHC class I downregulation was assessed as described above. The results of this analysis are shown on the graph.
    Figure Legend Snippet: MHC class I downregulation is independent of virus DNA replication. A. MHC class I downregulation is an early event in the virus lytic cycle. The lytic cycle was induced in Akata Bx1g cells by IgG cross-linking, and the levels of expression of immediate-early (BZLF1), early (EA-R-p85), and late (gp350/220) lytic cycle antigens were assessed by intracellular staining. In parallel, surface MHC class I was detected at each time point after induction. Percentages of cells positive for each of the antigens at the indicated time points are shown on the left y axis, whereas the relative MHC class I downregulation in GFP-positive cells compared with the GFP-negative cells is shown on the right y axis. B. MHC class I downregulation is independent of virus DNA replication and late gene expression. Akata Bx1g cells were induced by IgG cross-linking in the presence or absence of 0.2 mM acyclovir. At the indicated time points, cells were collected and stained for intracellular BZLF1 or gp350/220 expression. The numbers inside the dot plots indicate percentages of cells in the relevant quadrants. Mean fluorescence intensity of BZLF1-specific staining is shown for cells positive for both GFP and BZLF1. In parallel, samples were also stained for surface MHC class I, and the percentage of MHC class I downregulation was assessed as described above. The results of this analysis are shown on the graph.

    Techniques Used: Expressing, Staining, Fluorescence

    EBV lytic replication results in GFP expression and MHC class I downregulation in Akata Bx1g and AGS Bx1g cells. Akata cells carrying a recombinant EBV strain with the GFP gene inserted under the immediate-early CMV promoter into the virus genome (Akata Bx1g cells) or AGS gastric epithelial cells infected with the same virus (AGS Bx1g cells) were treated as described in Materials and Methods to induce the EBV lytic cycle. Staining with the indicated specific antibodies and fluorescence-activated cell sorter (FACS) analysis were performed as described in Materials and Methods. A. EBV replication, GFP expression, and MHC class I downregulation coincide in Akata Bx1g and AGS Bx1g cells. To induce EBV replication, cells were cultured either with F(ab′) 2 fragments specific to human IgG for the indicated periods of time (Akata Bx1g) or with TPA and sodium butyrate (AGS Bx1g) for 24 h, washed, and incubated in standard culture medium for the remaining time of induction. The four upper panels show intracellular staining for BZLF1. The lower panels show surface staining for MHC class I using W6/32 antibody. In both cases, anti-mouse APC-conjugated antibody was used to detect binding of primary antibodies. The R1 and R2 regions in the lower panels define two different cell populations, GFP − and GFP + cells, respectively, that were used for FACS analysis and cell sorting in this and all subsequent experiments. B. Signals used to activate the EBV lytic cycle in Akata or AGS cells induce overall upregulation of MHC class I, which is overridden by virus replication. Surface staining for MHC class I was done with W6/32 antibody and APC-conjugated anti-mouse secondary antibody. The upper panel shows the expression of MHC class I in the total population of either control (noninduced) or anti-IgG-treated (Akata-induced) or TPA-butyrate-treated (AGS-induced) cells. The four lower panels show either control or induced cells, each divided into GFP − and GFP + populations. The histograms are presented to illustrate the distribution of cells with different levels of MHC class I expression in cultures replicating the virus. C. Wild-type Akata cells downregulate MHC class I and MHC class II molecules upon induction of the EBV lytic cycle. Wild-type EBV-positive Akata cells were incubated with F(ab′) 2 fragments specific to human IgG for 24 h. Indirect intracellular staining for BZLF1 was followed by staining with directly conjugated MHC class I-, class II-, CD19-, or CD86-specific antibody. Cells were gated as BZLF1 positive (pos) or BZLF1 negative (neg). The geometric means of fluorescence intensities (Geo MFI) of the gated populations are shown. D. HLA-A, -B, and -C alleles are downregulated to similar extents during EBV replication. At 24 h after induction, the levels of surface expression of classical MHC class I molecules were assessed in Akata Bx1g cells with either a pan-MHC class I-specific antibody (W6/32) or antibodies specifically recognizing HLA-A, -B, or -C alleles. The geometric means of fluorescence intensity obtained with each of the indicated antibodies for GFP-positive cells are shown as percentages relative to those of anti-IgG-treated GFP-negative (latent) cells (the means and standard deviations of three experiments).
    Figure Legend Snippet: EBV lytic replication results in GFP expression and MHC class I downregulation in Akata Bx1g and AGS Bx1g cells. Akata cells carrying a recombinant EBV strain with the GFP gene inserted under the immediate-early CMV promoter into the virus genome (Akata Bx1g cells) or AGS gastric epithelial cells infected with the same virus (AGS Bx1g cells) were treated as described in Materials and Methods to induce the EBV lytic cycle. Staining with the indicated specific antibodies and fluorescence-activated cell sorter (FACS) analysis were performed as described in Materials and Methods. A. EBV replication, GFP expression, and MHC class I downregulation coincide in Akata Bx1g and AGS Bx1g cells. To induce EBV replication, cells were cultured either with F(ab′) 2 fragments specific to human IgG for the indicated periods of time (Akata Bx1g) or with TPA and sodium butyrate (AGS Bx1g) for 24 h, washed, and incubated in standard culture medium for the remaining time of induction. The four upper panels show intracellular staining for BZLF1. The lower panels show surface staining for MHC class I using W6/32 antibody. In both cases, anti-mouse APC-conjugated antibody was used to detect binding of primary antibodies. The R1 and R2 regions in the lower panels define two different cell populations, GFP − and GFP + cells, respectively, that were used for FACS analysis and cell sorting in this and all subsequent experiments. B. Signals used to activate the EBV lytic cycle in Akata or AGS cells induce overall upregulation of MHC class I, which is overridden by virus replication. Surface staining for MHC class I was done with W6/32 antibody and APC-conjugated anti-mouse secondary antibody. The upper panel shows the expression of MHC class I in the total population of either control (noninduced) or anti-IgG-treated (Akata-induced) or TPA-butyrate-treated (AGS-induced) cells. The four lower panels show either control or induced cells, each divided into GFP − and GFP + populations. The histograms are presented to illustrate the distribution of cells with different levels of MHC class I expression in cultures replicating the virus. C. Wild-type Akata cells downregulate MHC class I and MHC class II molecules upon induction of the EBV lytic cycle. Wild-type EBV-positive Akata cells were incubated with F(ab′) 2 fragments specific to human IgG for 24 h. Indirect intracellular staining for BZLF1 was followed by staining with directly conjugated MHC class I-, class II-, CD19-, or CD86-specific antibody. Cells were gated as BZLF1 positive (pos) or BZLF1 negative (neg). The geometric means of fluorescence intensities (Geo MFI) of the gated populations are shown. D. HLA-A, -B, and -C alleles are downregulated to similar extents during EBV replication. At 24 h after induction, the levels of surface expression of classical MHC class I molecules were assessed in Akata Bx1g cells with either a pan-MHC class I-specific antibody (W6/32) or antibodies specifically recognizing HLA-A, -B, or -C alleles. The geometric means of fluorescence intensity obtained with each of the indicated antibodies for GFP-positive cells are shown as percentages relative to those of anti-IgG-treated GFP-negative (latent) cells (the means and standard deviations of three experiments).

    Techniques Used: Expressing, Recombinant, Infection, Staining, Fluorescence, FACS, Cell Culture, Incubation, Binding Assay

    Free heavy chains accumulate during lytic replication. A. Total MHC class I heavy chains and β 2 m are downregulated in GFP-positive Akata Bx1g cells. Immunoblotting of total cell lysates of Akata Bx1g cells induced for 48 h with anti-IgG antibodies and sorted into GFP + (lytic) and GFP − (latent) cells. The heavy chain expression was assessed by two different antibodies: rαHC, a rabbit antiserum that recognizes heavy chains of HLA-A, -B, and -C, and HC10, a mouse monoclonal antibody specific for heavy chains of the HLA-B and -C loci. B. Both surface and total pools of assembled MHC class I molecules as well as β 2 m are downregulated during EBV replication while free MHC class I heavy chains accumulate in lytically infected cells. The upper panel shows density plots of Akata Bx1g cells 48 h after induction, surface stained with W6/32 (for assembled MHC class I), HC10 (for free MHC class I heavy chains), or TÜ99 (for β 2 m) antibodies obtained in one representative experiment. The lower graphs show the percentage of change in the expression of indicated molecules at the cell surface or in permeabilized GFP + cells relative to expression levels detected in GFP (latent) cells. The means and standard deviations of values obtained in 5 to 10 independent experiments are shown. FC, flow cytometry.
    Figure Legend Snippet: Free heavy chains accumulate during lytic replication. A. Total MHC class I heavy chains and β 2 m are downregulated in GFP-positive Akata Bx1g cells. Immunoblotting of total cell lysates of Akata Bx1g cells induced for 48 h with anti-IgG antibodies and sorted into GFP + (lytic) and GFP − (latent) cells. The heavy chain expression was assessed by two different antibodies: rαHC, a rabbit antiserum that recognizes heavy chains of HLA-A, -B, and -C, and HC10, a mouse monoclonal antibody specific for heavy chains of the HLA-B and -C loci. B. Both surface and total pools of assembled MHC class I molecules as well as β 2 m are downregulated during EBV replication while free MHC class I heavy chains accumulate in lytically infected cells. The upper panel shows density plots of Akata Bx1g cells 48 h after induction, surface stained with W6/32 (for assembled MHC class I), HC10 (for free MHC class I heavy chains), or TÜ99 (for β 2 m) antibodies obtained in one representative experiment. The lower graphs show the percentage of change in the expression of indicated molecules at the cell surface or in permeabilized GFP + cells relative to expression levels detected in GFP (latent) cells. The means and standard deviations of values obtained in 5 to 10 independent experiments are shown. FC, flow cytometry.

    Techniques Used: Expressing, Infection, Staining, Flow Cytometry, Cytometry

    14) Product Images from "Prevalence and Isotypic Complexity of the Anti-Chinese Hamster Ovary Host Cell Protein Antibodies in Normal Human Serum"

    Article Title: Prevalence and Isotypic Complexity of the Anti-Chinese Hamster Ovary Host Cell Protein Antibodies in Normal Human Serum

    Journal: The AAPS Journal

    doi: 10.1208/s12248-009-9165-5

    Confirmation of the specificity of the IgG4 type anti-CHO-HCP antibody in normal human serum ( HS ) samples—competitive inhibition specificity testing. Serum samples were pre-incubated with purified CHO-HCP at concentrations of 0, 10, 100, and 500 μg/ml,
    Figure Legend Snippet: Confirmation of the specificity of the IgG4 type anti-CHO-HCP antibody in normal human serum ( HS ) samples—competitive inhibition specificity testing. Serum samples were pre-incubated with purified CHO-HCP at concentrations of 0, 10, 100, and 500 μg/ml,

    Techniques Used: Inhibition, Incubation, Purification

    Identification of the IgG4 type reactivity in anti-CHO-HCP-positive serum samples using the biotin mouse anti-human IgG4 detector. IgG4 isotypic cut point OD is defined as two times the mean of the negative sample OD. Samples with OD values greater than
    Figure Legend Snippet: Identification of the IgG4 type reactivity in anti-CHO-HCP-positive serum samples using the biotin mouse anti-human IgG4 detector. IgG4 isotypic cut point OD is defined as two times the mean of the negative sample OD. Samples with OD values greater than

    Techniques Used:

    15) Product Images from "Curcumin alleviates immune-complex-mediated glomerulonephritis in factor-H-deficient mice"

    Article Title: Curcumin alleviates immune-complex-mediated glomerulonephritis in factor-H-deficient mice

    Journal: Immunology

    doi: 10.1111/imm.12079

    Curcumin (CMN) reduces IgG deposits in Complement factor H-deficient (CfH −/− ) mice with chronic serum sickness. Merged representative immunofluorescence photomicrographs are shown for IgG (blue), C3 (green) and C9 (red). C3 had the characteristic
    Figure Legend Snippet: Curcumin (CMN) reduces IgG deposits in Complement factor H-deficient (CfH −/− ) mice with chronic serum sickness. Merged representative immunofluorescence photomicrographs are shown for IgG (blue), C3 (green) and C9 (red). C3 had the characteristic

    Techniques Used: Mouse Assay, Immunofluorescence

    16) Product Images from "Interaction with the histone chaperone Vps75 promotes nuclear localization and HAT activity of Rtt109 in vivo"

    Article Title: Interaction with the histone chaperone Vps75 promotes nuclear localization and HAT activity of Rtt109 in vivo

    Journal: Traffic (Copenhagen, Denmark)

    doi: 10.1111/j.1600-0854.2011.01202.x

    Cytoplasmic Vps75 NLS mutants are functional in vivo. (A) Whole cell lysates from RTT109-TAP and RTT109-TAP vps75Δ strains transformed with the indicated Vps75 plasmids were western blotted using antibodies against IgG (for TAP), HA, and PGK1
    Figure Legend Snippet: Cytoplasmic Vps75 NLS mutants are functional in vivo. (A) Whole cell lysates from RTT109-TAP and RTT109-TAP vps75Δ strains transformed with the indicated Vps75 plasmids were western blotted using antibodies against IgG (for TAP), HA, and PGK1

    Techniques Used: Functional Assay, In Vivo, Transformation Assay, Western Blot

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    Article Snippet: .. AP-conjugated anti-mouse IgG (1:4,000; Sigma-Aldrich), IgG1 (1:4,000; MP Biomedicals, Solon, OH), or IgG2a (1:4,000; MP Biomedicals) was applied and incubated for 2 h at 37°C. .. Then p -nitrophenylphosphate (Bio-Rad) was applied, and the OD415 was measured after 20 min of incubation at 37°C by using a microplate reader (Bio-Rad).

    Inhibition:

    Article Title: Caspases switch off m6A RNA modification pathway to reactivate a ubiquitous human tumor virus
    Article Snippet: .. For caspase inhibition assay, cells were untreated or pretreated with caspase inhibitors (50 μM) for 1 hrs and then treated with IgG (1:200, Cat# 55087, MP Biomedicals) for additional 48 hrs. .. Cell Lysis and ImmunoblottingCell lysates were prepared in lysis buffer supplemented with protease inhibitors (Roche) as described previously.

    Article Title: Interferon regulatory factor 8 regulates caspase-1 expression to facilitate Epstein-Barr virus reactivation in response to B cell receptor stimulation and chemical induction
    Article Snippet: .. For caspase inhibition assay, Akata (EBV+) cells were untreated or pretreated with pan-caspase inhibitor for 1 hr and then treated with anti-IgG (1:200, Cat# 55087, MP Biomedicals) for additional 48 hrs. .. EBV reactivation in P3HR-1 cells was triggered by addition of TPA (20 ng/ml) and sodium butyrate (3 mM; Millipore, Cat# 19–137).

    other:

    Article Title: Monitoring of Anti-Hepatitis E Virus Antibody Seroconversion in Asymptomatically Infected Blood Donors: Systematic Comparison of Nine Commercial Anti-HEV IgM and IgG Assays
    Article Snippet: Consideration of seroconversion panels from donors 1 and 3 suggests that IgG antibodies seem to disappear using the MP-Bio (donor 1) or the Mikrogen, MP-Bio or Euroimmun assays (donor 3).

    Article Title: Caspases switch off m6A RNA modification pathway to reactivate a ubiquitous human tumor virus
    Article Snippet: Lytic Induction and Cell TreatmentFor lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 24 and 48 hrs.

    Avidin-Biotin Assay:

    Article Title: Mucosal IgA Responses in Healthy Adult Volunteers following Intranasal Spray Delivery of a Live Attenuated Measles Vaccine ▿
    Article Snippet: .. Measles virus-specific IgG and IgA in oral fluid and nasal washes were measured as described above, using biotin-labeled goat anti-IgA and anti-IgG (MP Biomedical, Solon, OH) diluted 1:2,000 in PBST followed by avidin peroxidase (Sigma, St. Louis, MO) diluted 1:400 in 1% bovine serum albumin (BSA) in PBS. .. Titers were calculated as the inverse of the dilution that produced an absorbance value of 0.2 above the blank value and are reported in ELISA units (EU)/ml.

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