Cellsens standard software version 1.6

Overnight cultures of cells with plasmids encoding fluorescent fusion proteins were diluted 1:100 in fresh LB medium with antibiotics and grown at 30°C with 0.005% arabinose for 4 h (unless otherwise stated). For imaging cells under no stress, a cell suspension (80 µl) was applied to a polylysine-coated slide, incubated for 15 min, and washed with LB medium (80 µl) before imaging. For imaging cells under stress (mostly E. coli), after 15 min of incubation, cells were washed, treated with LB containing the indicated stress-causing agent (80 µl), and incubated for 30 min before imaging. All slides for microscopy were prepared at room temperature. Stress-induced relocation was not dependent on immobilization by polylysine since it was also observed when stressors were directly added to broth cultures. For the E. coli temperature-sensitive mutants, after 30 min of stress exposure at room temperature, cells were incubated for another 30 min at the indicated temperature (30°C or 42°C) before microscopy. Images were acquired using an Olympus BX53 microscope, appropriate filters, and cellSens standard software (version 1.6) from Olympus and minimally processed using Adobe Photoshop 11.0.

For immunohistochemistry (IHC), the antigen retrieval step was performed with EDTA Buffer (1mM EDTA, 0.05% Tween 20, pH 8.0) in a pressure cooker. Endogenous peroxide activity was blocked by 3% peroxidase, and the slides were further blocked by 5% BSA to prevent nonspecific binding. Primary antibodies directed against PRKAR1A (Abcam, ab139695) incubated at an optimal dilution (1:100). The secondary antibody was donkey anti-rabbit IgG H&L (Abcam, ab6802, diluation 1:1000). For detection, the Polink-2 Plus horseradish peroxidase (HRP) Polymer Detection system (PV-9001; GBI Labs, Mukilteo, WA, USA) was used. Hematoxylin dye was used as counter stain. The slides were examined with an Olympus BX61 microscope with cell Sens Standard Software Version 1.6 (Olympus Corporation, Tokyo, Japan).