Journal: The Journal of Biological Chemistry

Article Title: C-tag TNF: a reporter system to study TNF shedding

doi: 10.1074/jbc.RA120.015248

Figure Lengend Snippet: C-tag TNF is shed in a similar fashion as WT TNF in HEK293T cells. A , Schematic overview of the C-tag TNF reporter. The C-tag is inserted in the TNF sequence before the ADAM17/10 cleavage site (between A76 and V77). Colons (:) represent identical amino acids, the plus sign (+) indicates a similar amino acid. B , Overview of possible detection methods for the C-tag TNF reporter. C , Immunoblotting of ADAM17 and ADAM10 in HEK293T cells of the indicated genotypes. * marks unspecific bands. D – E , HEK293T cells of the indicated genotypes were transfected with a control vector (pLI_mCherry) or with pLI_TNF ( D ) or pLI_C-tag TNF ( E ). Twenty-four hours after transfections, the expression of WT TNF and C-tag TNF was induced with doxycycline (1 µg/ml) in 2% FCS containing medium for 7 h before harvesting. Samples were analyzed via TNF ELISA and immunoblotting. memTNF in ( E ) was detected in cell lysates by the anti-C-tag nanobody. Data are represented as means ± S.E.M of 5 independent experiment or as representative images of 4 independent experiments. Uncropped images of the depicted immunoblots can be found in .

Article Snippet: Seven hours after transfection cells were treated with 1 μg/ml doxycycline for 36 h. On the day of sorting, 300 × 10 6 cells were detached from dishes, pooled and stained with CaptureSelect TM biotin anti-C-tag conjugate (Thermo Fisher Scientific, 1:185) and PE anti-human TNF (MAb11, Biolegend, 1:70) for 30 min on ice.

Techniques: Sequencing, Western Blot, Transfection, Control, Plasmid Preparation, Expressing, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: C-tag TNF: a reporter system to study TNF shedding

doi: 10.1074/jbc.RA120.015248

Figure Lengend Snippet: The C-tag TNF reporter can be used to detect TNF shedding by flow cytometry. HEK293T cells of indicated genotypes were transfected with pLI_BFP_C-tag TNF and protein expression induced by doxycycline for 36 h before analysis. A , Transfected cells were identified by BFP positivity and further analyzed for pro-TNF or C-tag positivity. B , Median fluorescence intensity (MFI) of pro-TNF and C-tag signals are reported in upper and lower panel, respectively. Data shown as means ± S.E.M of 3 independent experiments. C , ROC analysis of flow cytometry data shown in A to assess the discriminatory performance of pro-TNF and C-tag TNF staining between shedding and non-shedding (ADAM10 −/− × ADAM17 −/− ) cells. D , BFP positive cells were further separated in low, intermediate and high expression of pro-TNF before the analysis of C-tag signal in WT, ADAM17 −/− , ADAM10 −/− and ADAM10 −/− × ADAM17 −/− cells. E , MFI analysis of C-tag signal of histograms depicted in (D). Data shown as means ± S.E.M of 3 independent experiments. F , ROC analysis of flow cytometry data shown in ( D ) (upper, middle. and lower panel, respectively). Flow cytometry plots in ( A ) and ( D ) are representative of 3 independent experiments.

Article Snippet: Seven hours after transfection cells were treated with 1 μg/ml doxycycline for 36 h. On the day of sorting, 300 × 10 6 cells were detached from dishes, pooled and stained with CaptureSelect TM biotin anti-C-tag conjugate (Thermo Fisher Scientific, 1:185) and PE anti-human TNF (MAb11, Biolegend, 1:70) for 30 min on ice.

Techniques: Flow Cytometry, Transfection, Expressing, Fluorescence, Staining

Journal: The Journal of Biological Chemistry

Article Title: C-tag TNF: a reporter system to study TNF shedding

doi: 10.1074/jbc.RA120.015248

Figure Lengend Snippet: Live cell imaging of C-tag TNF shedding by confocal microscopy. HEK293T cells of the indicated genotypes were transiently transfected with pLI_C-tag TNF linker mCherry and pEF-BOS_nBFP, treated with doxycycline (1 µg/ml) for 25 h and imaged via confocal microscopy for a period of 7 h. A , Increasing positivity for C-tag TNF observed over time in WT HEK293T cells. Green = Alexa488 anti C-tag, blue = nBFP (nuclear BFP). White bar = 25 µm. B , C-tag and TNF linker mCherry signals observed over time by live cell imaging in cells of indicated genotypes. White bar = 25 µm. C – D , Quantification of cleaved TNF ( C ) and total TNF ( D ), respectively corresponding to Alexa488 anti-C-tag nanobody and mCherry signals. For each independent experiment, the average of signals from single cells in each channel was calculated. Data are presented as mean ± S.E.M of three independent experiments.

Article Snippet: Seven hours after transfection cells were treated with 1 μg/ml doxycycline for 36 h. On the day of sorting, 300 × 10 6 cells were detached from dishes, pooled and stained with CaptureSelect TM biotin anti-C-tag conjugate (Thermo Fisher Scientific, 1:185) and PE anti-human TNF (MAb11, Biolegend, 1:70) for 30 min on ice.

Techniques: Live Cell Imaging, Confocal Microscopy, Transfection

Journal: The Journal of Biological Chemistry

Article Title: C-tag TNF: a reporter system to study TNF shedding

doi: 10.1074/jbc.RA120.015248

Figure Lengend Snippet: A CRISPR/Cas9 genome-wide screen with the C-tag TNF reporter allows the identification of genetic factors involved in TNF shedding. A , HEK293T cells of depicted genotypes were transfected with pLI_C-tag TNF and protein expression induced for 36 h before analysis. Cells were pre-gated for high pro-TNF expression and then plotted for pro-TNF and C-tag TNF expression. A non-shedding population was identified by comparing the C-tag signal of WT cells with the signal obtained from ADAM17 −/− , ADAM10 −/− and ADAM10 −/− × ADAM17 −/− cells. One representative of 3 independent experiments. B , Schematic overview of the CRISPR/Cas9 screen strategy. C , Manhattan plot of hits obtained from the genetic screen. Data are shown as log2 of mean fold change (Mean LFC) of all gene specific gRNAs (max 4 gRNAs/gene) compared with unsorted control. The name of hits with mean LFC > 3 from the gene ranking list generated by PinAPL-Py analysis of screening data are reported. Additionally, the known regulator FURIN is depicted in the figure. D , WT, FURIN −/− and RHBDF2 −/− cells were transfected and doxycycline-treated as in ( A ). Cells were pre-gated on pro-TNF high expression and were further analyzed for C-tag signal. MFI of C-tag signal from pro-TNF high population is shown. Mean ± S.E.M of 3 independent experiments. One representative of 2 independent clones per genotype.

Article Snippet: Seven hours after transfection cells were treated with 1 μg/ml doxycycline for 36 h. On the day of sorting, 300 × 10 6 cells were detached from dishes, pooled and stained with CaptureSelect TM biotin anti-C-tag conjugate (Thermo Fisher Scientific, 1:185) and PE anti-human TNF (MAb11, Biolegend, 1:70) for 30 min on ice.

Techniques: CRISPR, Genome Wide, Transfection, Expressing, Control, Generated, Clone Assay