Overnight cultures of strains were
diluted 1:100 in 15 mL of 1× MM (1× M9 minimal media salts,
10 mM MgSO4, 1 mM CaCl2, 0.2% glucose, and 0.1%
Casamino acids, with 30 μg/mL kanamycin and 100 μg/mL
ampicillin for plasmid maintenance) and shaken at 37 °C. At mid
log phase, cultures were either untreated or ATc was added at a concentration
range from 0.005–0.2 μg/mL and cells were incubated at
37 o C. At 0, 10, and 60 min, 1 mL of culture was removed,
pelleted and resuspended in 100 μL of media. 50 μL was
mixed with 50 μL of 2× Laemmli buffer, denatured at 95
°C, and run on a 12% SDS-PAGE denaturing gel. The gel was transferred
to a polyvinylidene fluoride (PVDF) membrane using the iBlot Gel Transferring
System (Invitrogen). Membranes were probed with mouse anti-LexA (Santa
Cruz, sc-365999) 1:1000 or goat anti-MBP (NEB) 1:50 000, followed,
respectively, by horse radish peroxidase(HRP)-conjugated goat anti-mouse
or rabbit anti-goat (1:2000, Santa Cruz). Membranes were imaged on
a Amersham Imager 600 system after being exposed to Immobilon Western
HRP substrate (Millipore).
diluted 1:100 in 15 mL of 1× MM (1× M9 minimal media salts,
10 mM MgSO4, 1 mM CaCl2, 0.2% glucose, and 0.1%
Casamino acids, with 30 μg/mL kanamycin and 100 μg/mL
ampicillin for plasmid maintenance) and shaken at 37 °C. At mid
log phase, cultures were either untreated or ATc was added at a concentration
range from 0.005–0.2 μg/mL and cells were incubated at
37 o C. At 0, 10, and 60 min, 1 mL of culture was removed,
pelleted and resuspended in 100 μL of media. 50 μL was
mixed with 50 μL of 2× Laemmli buffer, denatured at 95
°C, and run on a 12% SDS-PAGE denaturing gel. The gel was transferred
to a polyvinylidene fluoride (PVDF) membrane using the iBlot Gel Transferring
System (Invitrogen). Membranes were probed with mouse anti-LexA (Santa
Cruz, sc-365999) 1:1000 or goat anti-MBP (NEB) 1:50 000, followed,
respectively, by horse radish peroxidase(HRP)-conjugated goat anti-mouse
or rabbit anti-goat (1:2000, Santa Cruz). Membranes were imaged on
a Amersham Imager 600 system after being exposed to Immobilon Western
HRP substrate (Millipore).