Figure 7. iTAP is essential for TACE maturation and function in primary cells and tissues from human and mouse.

(A). Schematic representation of the CRISPR targeting strategy to delete mouse Frmd8 (iTAP) gene using two guide RNAs flanking the first coding exon (exon 2). In the upper schematic of the Frmd8 locus, open boxes indicate non-coding exons whereas filled boxes indicate coding exons (B). Mouse embryonic fibroblasts (MEFs) were isolated from WT versus two independent iTAP KO E14.5 embryo littermates. The loss of iTAP at the protein level is shown by immunoblotting. (C). Mature TACE is diminished in iTAP KO MEFs. ConA-enriched lysates from MEFs isolated from WT versus iTAP KO embryos were deglycosylated as described previously. The transferrin receptor (TfR) is used as a loading control. (D). Mature TACE is depleted or diminished in TACE-relevant tissues from iTAP KO mice. ConA-enriched lysates from WT vs iTAP KO mouse tissues and bone marrow-derived macrophages, were deglycosylated as described previously. TACE was detected by western blot. The immature and mature species of TACE are indicated with white arrowheads and black arrowheads respectively, whereas red arrowheads denote the fully deglycosylated mature polypeptide. The experiment was performed twice with lysates isolated from tissues from two individual KO mice. (E). iTAP is essential for TACE physiological regulation in human primary cells. Isolated primary human peripheral blood mononuclear cells (PBMC) were differentiated into monocytes, then electroporated with the indicated siRNAs. Cells were then stimulated with the indicated concentrations of lipopolysaccharide (LPS). After 18 hr, the concentration of the cytokines TNF, IL-6 and IL-8 secreted into the supernatants, was measured by ELISA. The experiment was done three independent times and data from one representative experiment is shown. Data presented as mean ± standard error from triplicate measurements.

Figure 7—figure supplement 1. Mouse Frmd8/iTAP gene targeting via CRISPR.

(A). Schematic diagram showing the Frmd8 locus. Non-coding exons are denoted by open rectangles, whereas coding exons are shaded black. An enlarged area showing Frmd8 exons 1 and 2 is included to indicate the regions targeted by the CRISPR strategy. (B,C). Detailed maps of the WT and mutant loci of two different Frmd8 KO founder lines that were generated. A pair of gRNAs (indicated in red) was selected to engineer the deletion of the first coding exon of mouse Frmd8 as described in materials and methods. The PAM sequence and the theoretical Cas9 cut sites 3–4 bp from the PAM are indicated. The resulting founder animals were genotyped from tail biopsies and the identity of the lesions revealed by DNA sequencing. Two lines of animals (1 and 2) were established following germline transmission of the mutant alleles from individual founders. (B). KO line 1 contains a 478 bp deletion that removes exon 2 along with parts of introns 1 and 2. (C). KO line 2 contains a larger deletion of 625 bp that starts within the non-coding exon 1, deletes all of intron 1, exon 2 and part of intron 2, as shown. In both cases, the precise nucleotide sequence on the 5’ and 3’ boundaries of the mutant lines is indicated and aligned with the WT genomic sequence. The loss of iTAP at the protein level in both founder lines was confirmed by western blotting.