Bacterial pellets were resuspended in final sample buffer plus 0.2 M dithiothreitol (FSBS+DTT) and boiled for 15 min. At the time of loading, samples were normalized to the same number of cells by OD600. Protein samples were run on a 12.5% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAG) and transferred to a blotting membrane (Immobilon-P) with a wet mini trans-blot cell (Bio-Rad). Blots were blocked for an hour in Tris-buffered saline with Tween 20 and 5% skim milk and probed with rabbit anti-IscR (28 (link)), rabbit anti-YopD (gift from Alison Davis and Joan Mecsas), goat anti-YopE (Santa Cruz Biotechnology), rabbit anti-RpoA (gift from Melanie Marketon), and horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotech). Gels were imaged by Image Lab software (Bio-Rad).
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