with PBS and blocked in 200 ��l/well of 5 nonfat dry milk in PBS for 2 hours at room ASA-404 temperature. Full length Myc protein was diluted to 1 ng/��L in Ligustilide biological activity Buffer A . Compounds were serially diluted in 100 DMSO and then sequentially diluted 1:100 into the Myc solution before incubation for one hour at room temperature. For internal consistency, final DMSO concentrations were kept at 2. The blocked plate was washed 4x 200��l/well with Buffer A, before addition of 100 ��l of the Myc compound mixture. Plates were incubated for four hours at RT, and washed 4x 200 ��l Buffer A/well. Anti-Myc antibody was diluted 1:1000 in 5 nonfat dry milk in Buffer A. 100 ��L/well diluted primary antibody was added to each well and incubated for one hour at room temperature. Plates were washed 4x 200 ��l/well Buffer A. HRP-conjugated goat anti-rabbit antibody was diluted 1:5000 in 5 nonfat dry milk in Buffer A. 100 ��L/well diluted secondary antibody was added to each well and incubated for thirty minutes at room temperature. The plates were washed 4x 200 ��L/well Buffer A. 50 ��L/well of FEMTO chemiluminescent reagent was added, and luminescence was immediately read on a Victor X5 plate reader with a 0.1 sec integration time. IC50s were generated through non-linear fitting of data with prism . Human c-Myc containing the bHLH-LZ domain was cloned into a modified pET21a vector and then transformed into the BL21StarDE3 expression system, expressed, and purified at Cayman Chemical. The construct consists of an N-terminal 6xHis tag with a TEV cleavage site, c-Myc residues Asn352 to Ala439, and a GGCD C-terminal extension . Compounds were added to 200 ng c-Myc in reaction buffer and incubated for 60 minutes at room temperature. 350 ng His-tagged and GST-tagged full-length human Max protein was added, and the reaction was incubated for an additional 60 minutes. Finally, annealed double-stranded E-box containing DNA oligonucleotide with sequence 5��-GATCAGTTGACCACGTGGTCTGGG-3�� was added to a final concentration of 100 nM for twenty minutes incubation at room temperature. The final concentration of DMSO was kept constant at 2. The protein-DNA complexes were resolved
