After 2 days, the biofilms were washed once with sterile PBS before the addition of either 100µl of sterile PBS, PBS with reaction buffer, PBS with 40 Kuntz units/ml of DNase I, PBS with 40 Kuntz units/ml of heat inactivated DNase I, or PBS with reaction buffer and DNase I for 2h at 37°C.
After the initial 24-h incubation, the cells were washed twice with PBS before the addition of either fresh control media, prepared by mixing 2×-concentrated DMEM/FBS in a 1 1 ratio with water or experimental media, prepared by mixing 2×-concentrated DMEM/FBS in a 1 1 ratio with a BSA-SWNT dispersion containing ∼75 μg/mL of HiPco-SWNT material.
Oxygen consumption due to state 1 respiration was recorded for several minutes before the addition of either 5 mM glutamic acid plus 2.5 mM malic acid (complexes I, II, III, IV) or 5 mM succinic acid plus 2 mM rotenone (complexes II, III, IV), and oxygen consumption was again recorded.
For this, 1H-15N heteronuclear single quantum correlation (HSQC) spectra of the E2 UbcH5a were recorded before and after the addition of either RNF125stop99 or RNF125stop129.
Purified TOP1 was pre-incubated with GST or GST-BTBD2 for 20 min before addition of the DNA substrate, which was either an end-labeled 36 bp fragment containing a single strong TOP1 cleavage site [ 24], or a 161 bp PvuII-HindIII fragment of pBluescript (Stratagene) [ 25]).
The commercial feed was grounded into powder with a laboratory grinding machine and sieved before addition of the feed additives.
The samples were incubated for 5 min at 37 °C before addition of newly thawned microsomes.
The flask was evacuated and backfilled with argon, before addition of degassed EtOH (2.5 mL) and 33a (114 mg, 0.53 mmol).
