Adamtsl2 targeting strategy and validation of inactivation.

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Marcello Arsura, Min Wu, Gail E Sonenshein Immunity

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Adamtsl2 targeting strategy and validation of inactivation. Adamtsl2 targeting strategy and validation of inactivation. (A) Cartoon of the gene-targeting construct (top) and following Zp3-Cre excision of the neomycin (neo) cassette (bottom), which was performed to improve β-galactosidase staining. Primers used for genotyping (top) and for qRT-PCR (bottom) are indicated as arrows. (B) Adamtsl2 qRT-PCR was performed to verify Adamtsl2 deletion (means±s.d., n=3 per genotype) using two primer pairs with RNA extracted from lung tissue prior to Zp3-Cre mediated neo excision. Note that the expression of the RNA upstream of the targeting site (exon 2-3) is reduced, presumably due to the presence of the neo cassette, whereas downstream primers (exon 8-9) produce no product in Adamtsl2−/− lung. (C) Western blot of total lung protein from newborn mice probed with polyclonal antibody against the C-terminus of ADAMTSL2 (C3). 200 µg of total protein was loaded per lane. Two ADAMTSL2 bands (attributed to N-glycosylated and unmodified forms) at ∼150 kDa (arrows) are absent in protein extracts from Adamtsl2−/− lungs and are present at reduced levels in Adamtsl2+/− lungs (n=3 for WT and Adamtsl2−/−; n=2 for Adamtsl2+/−). (D) Conditioned medium from HEK293 cells secreting recombinant human ADAMTSL2 was used to confirm the reactivity and specificity of the anti-ADAMTSL2-C3 antibody. No reactivity was seen in the medium of empty-vector-transfected cells. (E) Cartoon of the domain organization of ADAMTSL1 (punctin-1) and ADAMTSL2. The localization of the anti-ADAMTSL2 C3 antibody epitope is indicated. (F) Western blot analysis of equal amounts of ADAMTSL1 (L1) and ADAMTSL2 (L2) detected with the anti-ADAMTSL2 antibody (L2C3, left-hand panel) or the anti-Myc (α-Myc) antibody (right-hand panel). Note that the anti-ADAMTSL2 antibody specifically recognized ADAMTSL2, whereas the α-Myc antibody recognized both proteins. The band at ∼50 kDa in the ADAMTSL2 lanes represents an ADAMTSL2 degradation product. En2SA, En2 splice acceptor; IRES, internal ribosome entry site; ns, non-specific bands. Dirk Hubmacher et al. Dis. Model. Mech. 2015;8:487-499 © 2015. Published by The Company of Biologists Ltd