Fig. 6

Adamts1 inhibits Notch signaling. a, b Time-course monitoring the expression levels of Notch target genes Hes1 and Hey1 in TA muscles isolated from wild-type mice before and after muscle injuries. Hes1 and Hey1 levels were quantified using RT-qPCR and the injured muscles were compared to the contralateral uninjured muscles (n = 5 mice per time point). c Representative western blot of lysates from primary myoblasts isolated from Tg and Wt mice. NICD=Notch1 intracellular domain (activated form of NOTCH1). Actin was used as a loading control. d Quantification of the levels of NICD protein in Tg (n = 7) compared to Wt (n = 8). e Quantification of Hes1 and Hey1 expression levels using RT-qPCR on primary satellite cells isolated from Wt (n = 3) and Tg (n = 3) mice by FACS. f Co-immunoprecipitation (IP) of ADAMTS1 and NOTCH1 from primary muscle tissue isolated from Tg mice using anti-HA (transgene has a hemagglutinin (HA) tag), anti-ADAMTS1 or IgG control antibodies. The transmembrane/intracellular fragment of NOTCH1 (TMIC) was immunoprecipitated specifically with ADAMTS1. NS is a nonspecific band. g Schematic of deletion and mutant Adamts1 expression constructs generated. h NOTCH1 co-IPs with ADAMTS1 and sequential ADAMTS1 deletion mutants map the NOTCH1 interaction domain to the metalloproteinase domain. Cont, control empty plasmid; FL, full-length Adamts1. i Western blots on cell lysates from co-cultured cells transfected with Notch1 and either Adamts1 (ADAMTS1) or E402Q Adamts1 (E402) or empty plasmid control (Control) expression plasmids. j (left) IP of processed myc-tagged NOTCH fragments from co-cultures with cells overexpressing Adamts1, Notch1 and Dll1 or empty vector controls. j (right) Quantification of the levels of NICD/TMIC fragments in response to co-culturing with Adamts1 overexpressing cells with and without cells expressing Dll1. P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent s.d. Statistical significance tested using paired t-tests