Fig. 4: Screening of HOIL-1L NZF inhibitors. | Cell Death & Disease

Fig. 4: Screening of HOIL-1L NZF inhibitors.

From: Synergistic involvement of the NZF domains of the LUBAC accessory subunits HOIL-1L and SHARPIN in the regulation of LUBAC function

Fig. 4

A, B Schematic illustration of the TR-FRET assay for detection of GST-tagged HOIL-1L NZF and His-tagged linear di-ubiquitin interactions (A). Counter TR-FRET assay with 6His-tagged GST protein (B). C, D, E Microscale thermophoresis (MST) assay of binding between HOIL-1L NZF and linear di-ubiquitin in the presence of compounds (C) and between compounds and linear di-ubiquitin (D). Correlation between C and D (E). F, G JR-GFP cells were treated with DMSO or the indicated compounds (10 μM) for 1 h and then stimulated with TNF-α (5 ng/mL) for 4 h (F) or TNF-α (5 ng/mL) plus CHX (20 μg/mL) for 6 h (G). Samples were analyzed by flow cytometry after Annexin V staining. Data are displayed as the mean fluorescence intensity (MFI) of GFP expression (F) and the frequency of Annexin V-positive cells (G). Data are shown as the mean ± s.d. of four independent experiments. P values are from a two-tailed Student’s t test.

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