Tran et al, supplemental material. P -cou 2 mM. O -cou 2 mM. pTZ. pTR. pTC. BS. P. MgCl 2. 2.5. 2.5. 1. 0.5. 0,25. 0. 0.5. 0,25. 0. PadR. probe. OD 600 = 1,6.
P-cou 2 mM
O-cou 2 mM
OD600 = 1,6
Fig. S1. Western-blot of purified polyclonal PadR antibodies on crude protein extracts from recombinant E. coli strains. Antibodies were produced and purified by Eurogentec, Belgium, from rabbits immunized with PadR purified from recombinant E. coli strain BL21pER. pTZ, control strain with the no-insert pTZ19R vector; pTR, pTZ19R harboring and expressing the padR gene; pTC, pTZ19 expressing padC; and BS, crude extract from the B. subtilis wild-type strain (see Table 1 and Fig. 4). Crude extracts were resolved by SDS-PAGE and proteins were electro-transferred onto a nitrocellulose membrane prior to Western-blotting.
Fig. S2. EMSA with the padC promoter probe and 1nM of PadR at different concentrations of MgCl2 from 0 to 2.5mM in the presence of 2 mM of p-coumaric (P-cou) or o-coumaric (O-cou) acid. P, padC promoter probe without protein. Effective inhibition of p-coumaric acid binding was detectable when MgCl2 concentrations were lower than 0.25mM. No effect was detected with o-coumaric acid even in the absence of MgCl2 (For EMSA conditions, refer to the legend of Fig. 6).