Journal: eLife

Article Title: iTAP, a novel iRhom interactor, controls TNF secretion by policing the stability of iRhom/TACE

doi: 10.7554/eLife.35032

Figure Lengend Snippet: ( A ). iTAP was knocked out in L929, RAW 264.7 and HEK 293ET cells using CRISPR. Lysates were immunoblotted with anti-iTAP antibodies. A small black arrowhead indicates iTAP protein whereas a non-specific band (white asterisk) serves as a loading control. ( B ). Glycoproteins from lysates isolated from the cells in ( A ) were enriched using concanavalin A-sepharose (conA) and TACE levels were assessed by western blot. Here and throughout, the immature form of TACE is indicated by a white arrow, whereas, the mature form is denoted by a black arrow. iRhom double KO MEFs were used as a reference and the transferrin receptor (TfR) as a loading control. Lower panels: densitometry in HEK 293ET. Left hand panel: Levels of immature TACE normalized to TfR. Right hand panel: levels of mature TACE as a relative proportion of immature TACE in WT and iTAP KO HEK 293ET. ( C,D ). Validation of mature and immature TACE detection in panels of WT versus iRhom2 DKO ( C ) or iTAP KO ( D ) cells, by deglycosylation. ConA enriched lysates from the cell lines in ( A ) were treated with endoglycosidase H (Endo-H; H; which cleaves ER-resident glycans only) and PNGase F (F; which cleaves both ER and post-ER glycans). Here and throughout: the immature TACE is indicated with white arrowheads; the black arrowhead denotes both glycosylated mature TACE and deglycosylated immature TACE respectively (which have similar electrophoretic mobility), whereas red arrowheads denote the fully deglycosylated, mature, TACE polypeptide. ( E ). iTAP expression restores the presence of mature TACE in iTAP KO cells. Lysates from WT or iTAP KO HEK 293ET stably expressing empty vector (-, EV) or human iTAP (+) were screened for mature TACE. Actin was used as a loading control. Middle and lower panels: densitometric analysis indicates that iTAP expression increases the levels of mature TACE but does not affect the levels of immature TACE. Middle panel: levels of mature TACE as a relative proportion of immature TACE in WT and KO upon iTAP or EV expression in WT and iTAP KO HEK 293ET clones. Lower panel: Levels of immature TACE after normalization to actin. ( F ). iTAP KO cells lack mature cell surface TACE. Left hand panel: RAW 264.7 WT or iTAP KO were surface-biotinylated in vivo and lysates were enriched for biotinylated proteins with neutravidin resin. Probing for TfR was used as a cell surface positive control protein whereas anti-p97 probing demonstrates that intracellular proteins were not labeled. Right hand panel: Cell surface biotinylated proteins were deglycosylated using Endo-H ( H ) or PNGase F ( F ). ConA enriched lysates were run as mobility controls, for immature and mature TACE. Blots were probed for TACE and for TfR as a control protein. ( G ). Loss of iTAP has no impact on the mature species of other ADAM metalloproteases. HEK 293ET WT or KO cells were transfected with the indicated panel of V5-tagged ADAMs. The lysates were deglycosylated as described above and Tubulin serves as a loading control. Throughout: Data are presented as mean ± standard deviation and represent three independent experiments. *=p ≤ 0.05, **=p ≤ 0.01, ***=p ≤ 0.001 and n.s. = non significant. 10.7554/eLife.35032.016 Figure 4—source data 1. iTAP KO cells are depleted in mature TACE levels. Densitometric analyses of mature/immature TACE levels. 10.7554/eLife.35032.017 Figure 4—source data 2. iTAP expression restores the presence of mature TACE in iTAP KO cells. Densitometric analyses of mature/immature TACE levels.

Article Snippet: Commercial assay or kit , Concanavalin A Agarose; conA , G-biosciences , 786–216 , .

Techniques: CRISPR, Control, Isolation, Western Blot, Biomarker Discovery, Expressing, Stable Transfection, Plasmid Preparation, Clone Assay, In Vivo, Positive Control, Labeling, Transfection, Standard Deviation

Journal: eLife

Article Title: iTAP, a novel iRhom interactor, controls TNF secretion by policing the stability of iRhom/TACE

doi: 10.7554/eLife.35032

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , Concanavalin A Agarose; conA , G-biosciences , 786–216 , .

Techniques: Generated, CRISPR, Isolation, Immunoprecipitation, Magnetic Beads, Negative Control, Transfection, Construct, Plasmid Preparation, Recombinant, Mutagenesis, Luciferase, Sequencing, TA Cloning, Enzyme-linked Immunosorbent Assay, Software

Journal: Vaccine

Article Title: Non-pathogenic Borrelia burgdorferi expressing Treponema pallidum TprK and Tp0435 antigens as a novel approach to evaluate syphilis vaccine candidates.

doi: 10.1016/j.vaccine.2019.02.022

Figure Lengend Snippet: Mice were immunized with 100 μl of non-infectious, bioluminescent B. burgdorferi strains B31HP- and B314-expressing Tp0435 mixed with an equal volume of 30 mg/ml D-luciferin substrate for B. burgdorferi codon-optimized luciferase encoded by the pJSB175-tp0435 plasmid. (A) Bioluminescence was observed within 10 mins of injection of the inoculum. (B) Loss of bioluminescence was detected within 24h, after delivering additional D-luciferin substrate at the cell injection site. (C) After a total of three immunizations, antibody titer against recombinant Tp0435 protein was determined by ELISA. B31HP-Tp0435: sera from mice immunized with Tp0435-expressing B31HP strain; B314-Tp0435: sera from mice immunized with Tp0435-expressing B314 strain. An antiserum against rTp0435 previously generated in mice using Freund’s adjuvant [33] was included as a control antiserum.

Article Snippet: Ten microliters/well of a sonicated suspension of B. burgdorferi was used as positive controls and 0.5 μg/well Concanavalin A (ICN Pharmaceuticals) as positive T-cell control.

Techniques: Expressing, Luciferase, Plasmid Preparation, Injection, Recombinant, Enzyme-linked Immunosorbent Assay, Generated, Adjuvant, Control

Journal: Vaccine

Article Title: Non-pathogenic Borrelia burgdorferi expressing Treponema pallidum TprK and Tp0435 antigens as a novel approach to evaluate syphilis vaccine candidates.

doi: 10.1016/j.vaccine.2019.02.022

Figure Lengend Snippet: (A) Reactivity against recombinant full-length Tp0435 (rTp0435, w/o signal peptide sequence, aa 25–156) was tested with sera obtained from rabbits prior to immunization (Prebleeds) and two weeks after each boost immunized with B. burgdorferi B31HP with vector control (rabbits R2654, R2652, R2651), and from rabbits immunized with B31HP strain expressing Tp0435 (rabbits R2649, R2650, R2653). (B) Reactivity against recombinant OspC (rOspC, aa 19–209) was tested in sera from all rabbits immunized with B. burgdorferi B31HP strains used in this study prior to immunization and two weeks after the 5th (and last) injection. Asterisks (*) indicate whether reactivity values after the 5th immunization are significantly higher (p≤0.05) as compared to the pre-immunization sera. Serum form a rabbit infected long term (>90 days) with the Nichols strain (IRS) was used as a positive control for T. pallidum antigens. (C) T-cell proliferation assay with splenic lymphocytes from rabbits immunized with B. burgdorferi expressing Tp0435 and unimmunized control rabbits using test antigens (rTp0435, rOspC, and rTp0136) and control antigens ConcanavalinA (ConA), sonicated B. burgdorferi (Bb), sonicated T. pallidum (Tp), and No antigen (No Ag). Bars represent the geometric means ± standard error of mean in quadruplicate experimental values for each antigen for three rabbits. Significant differences (p≤0.05) are indicated by an asterisk (*).

Article Snippet: Ten microliters/well of a sonicated suspension of B. burgdorferi was used as positive controls and 0.5 μg/well Concanavalin A (ICN Pharmaceuticals) as positive T-cell control.

Techniques: Recombinant, Sequencing, Plasmid Preparation, Control, Expressing, Injection, Infection, Positive Control, Proliferation Assay, Sonication

Journal: Vaccine

Article Title: Non-pathogenic Borrelia burgdorferi expressing Treponema pallidum TprK and Tp0435 antigens as a novel approach to evaluate syphilis vaccine candidates.

doi: 10.1016/j.vaccine.2019.02.022

Figure Lengend Snippet: RT-PCR analysis of tprK (panel to the left of marker) and ospC (panel right of marker) cDNA. Bb-TprK: B. burgdorferi expressing TprK; Tp: T. pallidum; Bb-VC: B. burgdorferi empty-vector control strain; NTC: no template control. gDNA: genomic DNA; RNA: total RNA; RNA+DNaseI: DNaseI-treated RNA.

Article Snippet: Ten microliters/well of a sonicated suspension of B. burgdorferi was used as positive controls and 0.5 μg/well Concanavalin A (ICN Pharmaceuticals) as positive T-cell control.

Techniques: Reverse Transcription Polymerase Chain Reaction, Marker, Expressing, Plasmid Preparation, Control

Journal: Vaccine

Article Title: Non-pathogenic Borrelia burgdorferi expressing Treponema pallidum TprK and Tp0435 antigens as a novel approach to evaluate syphilis vaccine candidates.

doi: 10.1016/j.vaccine.2019.02.022

Figure Lengend Snippet: TprK peptides identified by MS on B. burgdorferi B314 strain transformed with the pJB175- tprK vector

Article Snippet: Ten microliters/well of a sonicated suspension of B. burgdorferi was used as positive controls and 0.5 μg/well Concanavalin A (ICN Pharmaceuticals) as positive T-cell control.

Techniques: Transformation Assay

Journal: Vaccine

Article Title: Non-pathogenic Borrelia burgdorferi expressing Treponema pallidum TprK and Tp0435 antigens as a novel approach to evaluate syphilis vaccine candidates.

doi: 10.1016/j.vaccine.2019.02.022

Figure Lengend Snippet: (A) Absence of surface labelling of unpermeabilized empty vector containing B314 (Bb-VC) control strain by anti-TprK primary antiserum serum followed by detection with anti-rabbit Alexa fluor 488-conjugated secondary antibodies in contrast to positive surface staining of B. burgdorferi B314 expressing TprK (Bb-TprK) with anti-TprK antiserum. All spirochetes in the respective fields were imaged after simultaneous staining with DAPI that stains DNA. (B) Staining of all permeabilized and intact B. burgdorferi B314 strains with anti-OspC antibody was detected using anti-mouse TRITC-conjugated antibodies. (C) Integrity of these bacteria during IFA was confirmed by lack of staining of periplasmic flagella with monoclonal antibodies to flagellin followed by detection with anti-mouse TRITC-conjugated antibodies, unless cells were permeabilized by methanol treatment.

Article Snippet: Ten microliters/well of a sonicated suspension of B. burgdorferi was used as positive controls and 0.5 μg/well Concanavalin A (ICN Pharmaceuticals) as positive T-cell control.

Techniques: Plasmid Preparation, Control, Staining, Expressing, Bacteria, Bioprocessing

Journal: Vaccine

Article Title: Non-pathogenic Borrelia burgdorferi expressing Treponema pallidum TprK and Tp0435 antigens as a novel approach to evaluate syphilis vaccine candidates.

doi: 10.1016/j.vaccine.2019.02.022

Figure Lengend Snippet: (A) Reactivity of antisera from immunized rabbits against rTprK (w/o signal peptide sequence, aa 37–273) was tested in sera collected prior to immunization and two weeks after each boost from rabbits immunized with B. burgdorferi B314 vector control (rabbits R2654, R2652, R2651) and with B. burgdorferi B314 expressing TprK (rabbits R8677, R8679, and R8680). (B) Reactivity of immunized rabbit sera against recombinant OspC (aa 19–209) was tested two weeks after the 5th (and last) injection and compared with the pre-immunization sera. Bars represent the mean OD readings after background (from no antigen wells) subtraction. Asterisks (*) indicate whether reactivity values after the 5th immunization are significantly higher (p≤0.05) than the pre-immunization rabbits sera.

Article Snippet: Ten microliters/well of a sonicated suspension of B. burgdorferi was used as positive controls and 0.5 μg/well Concanavalin A (ICN Pharmaceuticals) as positive T-cell control.

Techniques: Sequencing, Plasmid Preparation, Control, Expressing, Recombinant, Injection

Journal: Vaccine

Article Title: Non-pathogenic Borrelia burgdorferi expressing Treponema pallidum TprK and Tp0435 antigens as a novel approach to evaluate syphilis vaccine candidates.

doi: 10.1016/j.vaccine.2019.02.022

Figure Lengend Snippet: (A) Sera collected from rabbits immunized with B. burgdorferi expressing TprK were used in ELISA to determine which sequences of TprK elicited a humoral response in each animal. Marked peptides contain conserved (filled arrows) and variable (open arrows) sequences predicted to be surface exposed. rTprK was used as a positive control. (B) Reactivity of sera from immunized animals (R8677, R8679, and R8680) to refolded TprK (refTprK) and TprK recombinant antigen denatured using 6M urea (denTprK). Bars represent the mean OD readings after background (from no antigen wells) subtraction. Asterisk (*) indicates significant increase in cumulative reactivity (p≤0.05).

Article Snippet: Ten microliters/well of a sonicated suspension of B. burgdorferi was used as positive controls and 0.5 μg/well Concanavalin A (ICN Pharmaceuticals) as positive T-cell control.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Positive Control, Recombinant