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: Scientific Reports

Article Title: TUSC3 Loss Alters the ER Stress Response and Accelerates Prostate Cancer Growth in vivo

doi: 10.1038/srep03739

Figure Lengend Snippet: (a) shTUSC3 and control (scrambled shRNA) PC3 and DU145 cells, respectively, were evaluated using transmission electron microscopy in ultrathin sections. Only cells with intact plasma membrane, nucleus, nucleolus, mitochondria, ER and Golgi apparatus were included into the analysis. We examined 20 cells of each cell type. Arrows indicate branching or dilatation of ER cisternae. Scale bars correspond to 2 μm. TUSC3 silencing promotes prostate cancer xenograft growth. (b) shTUSC3 and control (scrambled shRNA) PC3 and DU145 cells, respectively, were injected into the flanks of 6–8-week old male nude mice. Tumor growth was monitored and is shown as mean tumor size (+/− SEM). (c) Representative tumor formed by the TUSC3 silenced PC3 xenograft in the right flank of a Foxn1 nu/nu mouse (top left). Sustained downregulation of TUSC3 in mouse tumors was analyzed by qRT-PCR (top right) and immunohistochemistry (TUSC3 IHC, middle graphs). Major differences in N-glycosylation in mouse tumors were not identified by Concanavalin A lectin histochemistry (ConA lectin, bottom graphs).

Article Snippet: Lectin histochemistry and cytochemistry was performed using Concanavalin A (ConA) HRP conjugate in a concentration of 1:40 in PBS (USBiological, Swampscott, MA, USA) overnight at 4°C, followed by 2xPBS washes and visualized with Dako Liquid DAB + Substrate-Chromogen System (K3468).

Techniques: Control, shRNA, Transmission Assay, Electron Microscopy, Clinical Proteomics, Membrane, Injection, Quantitative RT-PCR, Immunohistochemistry, Glycoproteomics