Journal: Nucleic Acids Research

Article Title: Co-regulated expression of alpha and beta mRNAs encoding HLA-DR surface heterodimers is mediated by the MHCII RNA operon

doi: 10.1093/nar/gkt059

Figure Lengend Snippet: Modulation of MHCII expression. ( A ) The graphs illustrate the time course (0, 30, 48 and 72 h) of DRA and DRB mRNAs fold variation in M14 cells measured by qRT–PCR after 24 h of EBP1 and NF90 proteins silencing and subsequent transfections of indicated plasmids. In details, subpanels show quantification of two messengers after silencing of EBP1 (I) and NF90 (II) and after silencing with siCtrl followed by p5DRA3 (III) and p5DRB3 (IV) transfections. Silencing with siEBP1 followed by p5DRA3 (V) and p5DRB3 (VI) transfections, and knockdown with siNF90 followed by p5DRA3 (VII) and p5DRB3 (VIII) transfections were shown. ( B ) (I) Cytofluorimetric analysis of HLA-DR surface expression in HeLa cells 48 h after transfection with indicated amount of pSVK3, p5DRA3 and p5DRB3 reported as fold change of MFI. (II) DRA and DRB mRNAs variation measured by qRT–PCR in cells were analysed in the same experimental conditions. In the case of DRB mRNA, P -value is calculated with respect to the maximum amount of p5DRA3 used; for DRA mRNA, P -value is calculated with respect to the maximum amount of p5DRB3 transfected.

Article Snippet: The presence of NF90 protein was verified by western blot analysis with specific anti-DRBP76 (anti-double stranded RNA binding protein 76 or anti-NF90) antibody (BD Biosciences), whereas the presence of EBP1 protein was confirmed by anti-FLAG-M2 (Sigma) and anti-EBP1 (Abcam) antibodies.

Techniques: Expressing, Quantitative RT-PCR, Transfection

Journal: Nucleic Acids Research

Article Title: Co-regulated expression of alpha and beta mRNAs encoding HLA-DR surface heterodimers is mediated by the MHCII RNA operon

doi: 10.1093/nar/gkt059

Figure Lengend Snippet: Analysis of interactions of MHCII RNP components. ( A ) REMSAs experiments performed using 5DRA (lane 1) riboprobe; lane 2 shows the digestion with T1 RNase. Lane 3 shows the band of interaction of M14 extract with 5DRA. Competition experiments of 5DRA binding were performed using 0.5, 1 and 2.5 µg of cold 5DRB (lanes 4–6), 3DRA (lanes 7–9) and 3DRB (lanes 10–12). ( B ) REMSAs experiments performed using 5DRB (lane 1) riboprobe; lane 2 shows the digestion with T1 RNase. Lane 3 shows band of interaction of M14 extract with 5DRB. Competition experiments of 5DRB binding were performed using 0.5, 1 and 2.5 µg of cold 5DRA (lanes 4–6), 3DRB (lanes 7–9) and 3DRA (lanes 10–12). ( C ) REMSAs experiments performed using 5DRA (on the left) and 5DRB (on the right) riboprobes. Lane 1 shows the interaction with rEBP1, lane 2 with rNF90 and lane 3 shows band of binding with M14 extract, whereas lanes 4–6 show the supershift of cytoplasmic complex with anti-DRBP76 in binding reaction containing 0.25, 1.25 and 2.5 µg of anti-DRBP76 antibody. No supershift was evident in the presence of 2.5 µg of control IgG (lane 7). Lane 8 shows the interaction of 5DRB with rEBP1, lane 9 demonstrates the absence of binding with rNF90, lanes 10 shows band of interaction of M14 extract, whereas no supershift of cytoplasmic complex with anti-DRBP76 was found in binding reaction containing 0.25, 1.25 and 2.5 µg of anti-DRBP76 antibody (lanes 11–13), similar to that observed in the presence of 2.5 µg of control IgG (lane 14). ( D ) Western blot analysis of biotin pull-down assay carried by using 5DRA, 5DRB, 3DRA, 3DRB and UBC biotinylated riboprobes, after incubation whit cytoplasmic lysate from M14 cells. The top panel represents the immunoblot performed by anti-NF90, whereas the middle and the bottom panels correspond to the western carried out by anti-FLAG and anti-EBP1, respectively, able to detect EBP1 protein. Molecular weights are indicated.

Article Snippet: The presence of NF90 protein was verified by western blot analysis with specific anti-DRBP76 (anti-double stranded RNA binding protein 76 or anti-NF90) antibody (BD Biosciences), whereas the presence of EBP1 protein was confirmed by anti-FLAG-M2 (Sigma) and anti-EBP1 (Abcam) antibodies.

Techniques: Binding Assay, Western Blot, Pull Down Assay, Incubation

Journal: Nucleic Acids Research

Article Title: Co-regulated expression of alpha and beta mRNAs encoding HLA-DR surface heterodimers is mediated by the MHCII RNA operon

doi: 10.1093/nar/gkt059

Figure Lengend Snippet: RNA secondary structure motifs. ( A ) Locations of the common secondary structure motifs between the 5′- and 3′-UTRs of the same RNA to form an enclosed loop [(A) HLA-DRA and (C) HLA-DRB] and between the two different RNAs to form a heterogenic complex [(C) DRA-5′-UTR to DRB-3′-UTR and (D) DRB-5′-UTR to DRA-3′-UTR]. In the HLA-DRB 5′-UTR, there are two overlapping structures that are unlikely to be able to co-exist; therefore, only one at any one time can act as a cis -acting signal. ( B ) The individual RNA secondary structure motifs arranged in matching pairs. Each motif is locally stable and with a >50% sequence identity. ( C ) Possible binding modes between the common secondary structure motifs within the RNP containing EBP1 and NF90. The binding modes are between the 5′- and 3′-UTR of the same RNA molecule and between the UTRs of different RNAs.

Article Snippet: The presence of NF90 protein was verified by western blot analysis with specific anti-DRBP76 (anti-double stranded RNA binding protein 76 or anti-NF90) antibody (BD Biosciences), whereas the presence of EBP1 protein was confirmed by anti-FLAG-M2 (Sigma) and anti-EBP1 (Abcam) antibodies.

Techniques: Sequencing, Binding Assay

Journal: Frontiers in Plant Science

Article Title: CRISPR/Cas9 suppression of OsAT10, a rice BAHD acyltransferase, reduces p-coumaric acid incorporation into arabinoxylan without increasing saccharification

doi: 10.3389/fpls.2022.926300

Figure Lengend Snippet: Constructs used and targets for CRISPR/Cas within OsAT10. (A) Schematic presentation of the T-DNA structure in the vectors utilized in this study. The expression of the sgRNA scaffold is driven by the rice U3 promoter (OsU3), expression of Cas9 by the rice ubiquitin promoter (P Osubi) and expression of hygromycin selection marker (HPT) by Maize ubiquitin promoter. Abbreviations: NLS, nuclear localization signal; Tnos, Nopaline terminator; T CaMV, Cauliflower Mosaic Virus (CaMV) 35S terminator; LB, Left border; RB, right border. (B) Indels present in the original lines, KO lines selected are marked and PAM is underlined. (C) Schematic of the OsAT10 gene structure and target site. The intron is marked with gray and the two exons are shown with thick black lines. The location of the two crisprs are marked.

Article Snippet: Initially, the sgRNA expression cassette was cloned into vector pJIT163-2NLSCas9 (Addgene: Plasmid #53064; ), while simultaneously adding a ubiquitin promoter to the Cas9 gene.

Techniques: Construct, CRISPR, Expressing, Selection, Marker

Journal: Scientific Reports

Article Title: Multi-level glyco-engineering techniques to generate IgG with defined Fc-glycans

doi: 10.1038/srep36964

Figure Lengend Snippet: ( a ) A schematic model of the bi-antennary glycan as present on IgG Fc at position N297. ( b ) NanoLC-ESI-MS spectrum of IgG1 produced in HEK cells and ( c ) human plasma-derived IVIg Kiovig IVIg (data obtained from ref. ), exhibit a similar glycosylation pattern for the Fc glycan for each direct glycan trait. ( d ) The similarity is also evident when visualized as calculated derived glycan traits, IgG glycosylation profile of either HEK freestyle produced IgG (this study n = 28) or IVIg (n = 61, data obtained from ref. ), Error bars indicate standard deviation (SD), **** denotes a statistical significance of p ≤ 0.0001 tested by unpaired t-test.

Article Snippet: Antibodies were purified from the supernatant on a protein A (WT IgG1) HiTrap HP column (GE Life Sciences, 29-0485-76) and dialyzed against phosphate buffered saline (PBS) overnight.

Techniques: Produced, Derivative Assay, Standard Deviation

Journal: Scientific Reports

Article Title: Multi-level glyco-engineering techniques to generate IgG with defined Fc-glycans

doi: 10.1038/srep36964

Figure Lengend Snippet: ( a ) Flowchart of IgG production in HEK freestyle cell line and glycan analysis by mass spectrometry, illustrating the 4 steps of IgG production on a time scale, with arrows indicating the glyco-engineering methods employed at each step. After the in vitro ST6GALT treatment, an additional purification step was required (dashed line with arrow). ( b ) To analyse the glycosylation profile, the resulting IgG was digested with trypsin and the glycopeptides encompassing the N -glycan were analysed by mass spectrometry.

Article Snippet: Antibodies were purified from the supernatant on a protein A (WT IgG1) HiTrap HP column (GE Life Sciences, 29-0485-76) and dialyzed against phosphate buffered saline (PBS) overnight.

Techniques: Mass Spectrometry, In Vitro, Purification

Journal: Scientific Reports

Article Title: Multi-level glyco-engineering techniques to generate IgG with defined Fc-glycans

doi: 10.1038/srep36964

Figure Lengend Snippet: ( a ) The fucose level of IgG1 N297, produced by transfection of IgG heavy and light chain vector in combination with co-transfection of RMD vector. ( b ) or with addition of 2FF or a combination of 2FF and co-transfection with 5% RMD. ( c ) Effect of 2FF addition on other derived glycosylation traits (bisection, galactosylation, sialylation). ( d ) NanoLC-ESI-MS spectrum of IgG produced with 0.4 mM 2FF. The data represents means and SD of three combined independent experiments, * and **** denote a statistical significance of p ≤ 0.05 and p ≤ 0.0001, respectively, as tested by one-way ANOVA using Dunnett’s multiple comparisons test comparing untreated cells with treated. The vertical dotted lines in ( b , c ) represent the designated optimal concentrations of 2FF.

Article Snippet: Antibodies were purified from the supernatant on a protein A (WT IgG1) HiTrap HP column (GE Life Sciences, 29-0485-76) and dialyzed against phosphate buffered saline (PBS) overnight.

Techniques: Produced, Transfection, Plasmid Preparation, Cotransfection, Derivative Assay

Journal: Scientific Reports

Article Title: Multi-level glyco-engineering techniques to generate IgG with defined Fc-glycans

doi: 10.1038/srep36964

Figure Lengend Snippet: ( a ) The level of bisecting GlcNAc (bisection) on IgG1 N297 after co-transfection of IgG heavy and light chain vectors with GNTIII vector. ( b ) The effect of GNTIII co-transfection on other derived glycosylation traits (fucosylation, galactosylation, sialylation). ( c ) NanoLC-ESI-MS spectrum of IgG produced with 1% GNTIII co-transfection. The data represents means and SD from two independent experiments carried out in an identical fashion, representative of 3 independent experiments *, **, *** and **** denote a statistical significance of p ≤ 0.05, p ≤ 0.01, p ≤ 0.001 and p ≤ 0.0001, respectively, as tested by one-way ANOVA using Dunnett’s multiple comparisons test comparing untreated cells with treated. The vertical dotted lines in ( a , b ) represent the designated optimal concentrations of GNTIII.

Article Snippet: Antibodies were purified from the supernatant on a protein A (WT IgG1) HiTrap HP column (GE Life Sciences, 29-0485-76) and dialyzed against phosphate buffered saline (PBS) overnight.

Techniques: Cotransfection, Plasmid Preparation, Derivative Assay, Produced

Journal: Scientific Reports

Article Title: Multi-level glyco-engineering techniques to generate IgG with defined Fc-glycans

doi: 10.1038/srep36964

Figure Lengend Snippet: ( a ) The galactosylation level of IgG1 N297 produced with addition of 2FG. ( b ) Effect of 2FG addition on other derived glycosylation traits (fucosylation, bisection, sialylation). ( c ) NanoLC-ESI-MS spectrum of IgG produced with 1 mM 2FG. The data represents means and SD of four combined independent experiments, *, **, *** and **** denote a statistical significance of p ≤ 0.05, p ≤ 0.01, p ≤ 0.001 and p ≤ 0.0001, respectively, as tested by one-way ANOVA using Dunnett’s multiple comparisons test comparing untreated cells with treated. The vertical dotted lines in ( a , b ) represent the designated optimal concentrations of 2FG.

Article Snippet: Antibodies were purified from the supernatant on a protein A (WT IgG1) HiTrap HP column (GE Life Sciences, 29-0485-76) and dialyzed against phosphate buffered saline (PBS) overnight.

Techniques: Produced, Derivative Assay

Journal: Scientific Reports

Article Title: Multi-level glyco-engineering techniques to generate IgG with defined Fc-glycans

doi: 10.1038/srep36964

Figure Lengend Snippet: ( a ) The galactosylation level of IgG1 N297 after co-transfection of IgG heavy and light chain vectors with B4GALT1 or B4GALT2 vectors, and ( b ) after addition of D-galactose or in combination with co-transfected 1% B4GALT1, or 1% B4GALT2. ( c ) The effect of B4GALT1 co-transfection or ( d ) or D-galactose addition on the other derived glycosylation traits (fucosylation, bisection, sialylation). ( e ) NanoLC-ESI-MS spectrum of IgG produced with 1% B4GALT1 co-transfection and 1 mM D-galactose addition. The data represents means and SD from two independent experiments carried out in an identical fashion, representative of 3 independent experiments, *, *** and **** denote a statistical significance of p ≤ 0.05, p ≤ 0.001 and p ≤ 0.0001, respectively, as tested by one-way ANOVA using Dunnett’s multiple comparisons test comparing untreated cells with treated. The vertical dotted lines in ( a , b ) represent the designated optimal concentrations of B4GALT1, and in ( c , d ) the optimal concentrations of D-Galactose.

Article Snippet: Antibodies were purified from the supernatant on a protein A (WT IgG1) HiTrap HP column (GE Life Sciences, 29-0485-76) and dialyzed against phosphate buffered saline (PBS) overnight.

Techniques: Cotransfection, Transfection, Derivative Assay, Produced

Journal: Scientific Reports

Article Title: Multi-level glyco-engineering techniques to generate IgG with defined Fc-glycans

doi: 10.1038/srep36964

Figure Lengend Snippet: ( a ) The sialylation level of IgG1 N297 after co-transfection of IgG heavy and light chain vectors with 1% B4GALT1 and addition of 5 mM D-galactose to increase galactosylation, in combination with increasing amount of co-transfected ST6GALT. N = 2 ( b ) Effect of ST6GALT co-transfection on other derived glycosylation traits. N = 2 ( c ) In vitro sialylation by ST6GALT of highly galactosylated and sialylated IgG1. Before treatment N = 3, after treatment N = 7 ( d ) NanoLC-ESI-MS spectrum of IgG produced with the identified optimal co-transfection additions of B4GALT1 (1%) and ST6GALT (2,5%) with the addition 1 mM D-galactose, and ( e ) spectrum of in vitro sialylated IgG produced in ( d ). The data represents means and SD, **** denotes a statistical significance of p ≤ 0.0001 tested by one-way ANOVA using Dunnett’s multiple comparisons test ( a , b ) or unpaired t-test ( c ) comparing untreated cells or IgG respectively with treated. The vertical dotted lines in ( a,b ) represent the designated optimal concentrations of ST6GAL.

Article Snippet: Antibodies were purified from the supernatant on a protein A (WT IgG1) HiTrap HP column (GE Life Sciences, 29-0485-76) and dialyzed against phosphate buffered saline (PBS) overnight.

Techniques: Cotransfection, Transfection, Derivative Assay, In Vitro, Produced