Wet mini trans blot cell

In parallel, after the supernatant was collected, bacterial pellets were resuspended in FSB plus 20% DTT. Pellet samples were boiled for 15 min. At the time of loading, samples were normalized to the same number of cells. Cytosolic and secreted protein samples were run on a 12.5% SDS-PAGE gel 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 the goat anti-YopE antibodies (Santa Cruz Biotechnology), rabbit anti-YopD (gift from Alison Davis and Joan Mecsas), rabbit anti-RpoA (gift from Melanie Marketon), rabbit anti-LcrF [35 (link)], rabbit anti-IscR [25 (link)], and horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotech). Following visualization, quantification of the bands was performed with Image Lab software (Bio-Rad).

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).

Cell pellets were collected, resuspended in FSB plus 0.2 M DTT, and boiled for fifteen minutes. At the time of loading, supernatants and cell pellets were normalized to the same number of cells. After separation on a 12.5% SDS-PAGE gel, proteins were transferred onto 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 the rabbit anti-RpoA (gift from Melanie Marketon), rabbit anti-LcrF (gift from Gregory Plano), rabbit anti-IscR [44 (link)], rabbit anti-YmoA (gift from Gregory Plano), rabbit anti H-NS (gift from Robert Landick), mouse M2 anti-FLAG (Sigma), goat anti-YopE (Santa Cruz Biotech), and horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotech). Following visualization, quantification of the bands was performed with Image Lab software (Bio-Rad).