Immunoblotting for CdiA protein

Cells were collected by centrifugation and frozen at –80 °C. Frozen cells were resuspended in urea-lysis buffer [50% urea, 150 mM NaCl, 20 mM Tris-HCl (pH 8.0)] and subjected a freeze-thaw cycle to extract proteins. Urea-soluble proteins were resolved by SDS-PAGE on Tris-tricine 6% polyacrylamide gels run at 100 V (constant) for 3 h. Gels were soaked for 15 min in 25 mM Tris, 192 mM glycine (pH 8.6), 10% methanol, then electroblotted to low-fluorescence PVDF membranes using a semi-dry transfer apparatus at 17 V (constant) for 1 h. Membranes were blocked with 4% non-fat milk in 1× PBS for 1 h at room temperature and incubated with primary antibodies in 0.1% non-fat milk, 1× PBS overnight at 4 °C. Rabbit polyclonal antisera to the TPS domain of CdiA (residues Val33 - Gly285) were generated by Cocalico Biologicals Inc. (Reamstown, PA) as described by ref. 67 (link). Anti-TPS antisera were used at a 1:10,000 dilution to detect CdiA proteins by immunoblotting. Blots were incubated with 800CW-conjugated goat anti-rabbit IgG (1:40,000 dilution, LI-COR) in 0.1% non-fat milk in PBS. Immunoblots were visualized with an LI-COR Odyssey infrared imager. All uncropped and unprocessed infrared imager scans are provided in the Source Data files. Samples of the polyclonal anti-CdiA antisera are available from the corresponding author upon request.