Quikchange 2 protocol

All wildtype and mutagenized enhancer-reporter transgenic constructs were made using the lacZ reporter vector pRVV54 as an acceptor vector [49 (link)]. Coordinates of the genomic fragments PCR-amplified in the enhancer bashing experiments are listed in S1 and S5 Tables. The ΦC31 system was used for transgenesis and plasmids were introduced in landing sites 51D or 86Fa [50 (link)].
Site-directed mutagenesis of the vnE and rhoE enhancers was performed according to the QuikChange II protocol (Agilent Technologies). vnE and rhoE enhancers were first introduced in pBluescript SK+ vector for site-directed mutagenesis and the resulting mutated enhancers were consequently transferred to pRVV54 for in vivo analysis in the fruit fly. Primers used for mutagenizing of putative binding site are listed in S3 Table.
Plasmids for recombinant protein production were made by introducing cDNA sequences into pET21 series vectors (Novagen-EMD Millipore) and their derivatives, resulting in C-terminally tagged His proteins. Primers used to generate Dll-His (full-length Dll), Sp1^Zn-finger-His (only the Zn-finger domains; used for confirming in vitro binding to Sp1 sites), Sp1^424AA-His (used to examine cooperativity with Dll), Mad^MH1-His (only the MH1 domain) and Pan^HMG-His (only the HMG domain) vectors are listed in S2 Table.

Recombinant proteins for use in in vitro assays were expressed using auto-induction with BL21 Escherichia coli (Novagen) transformed with a modified pET24b( + ) vector (Novagen) containing a tac promoter cloned at BglII and XbaI restriction sites^{45 (link), 46 (link)}. Ch-CPN gene was inserted at NdeI and XhoI restriction sites. Cells were collected by centrifugation at 5,000 × g for 10 min at room temperature, then resuspended (5% wt vol⁻¹) in 50 mM NaH₂PO₄ (pH 8.0) and 300 mM NaCl in the presence of 1 mM phenylmethylsulfonyl fluoride followed by lysis via French Press. Proteins used in ATPase assays were initially resuspended and lysed in HEPES buffer (50 mM HEPES (pH 8.0) and 100 mM NaCl) to eliminate background phosphate. The lysate was centrifuged at 20,000 × g for 20 min at room temperature. The supernatant was heated at 50 °C for 30 min followed by additional centrifugation at 20,000 × g for 20 min to remove aggregation. Heat-treated and clarified supernatant was loaded onto anion exchange High Q column (Bio-Rad) and eluted in a linear gradient up to 1 M NaCl. Protein concentrations were determined by Coomassie Plus (Bradford) reagent (Pierce) at 595 nm. Site-directed mutagenesis was performed following the QuikChange II protocol (Agilent Technologies). The primer sequences used for constructing expression plasmids are described in Supplementary Table 3.