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