Cell Lysate Immunoblotting Protocol

The protocols used for cell lysate preparation and immunoblotting were described previously [42 (link)]. Briefly, 1x10⁸ bacteria were mixed with 100 μl of SDS-PAGE sample buffer (45 mM Tris-Cl, pH 6.8, 10% glycerol, and 1% SDS in the presence or absence of 5% β-mercaptoethanol or 50 mM DTT). A 10 μl sample of cell lysate or 20–40 μl of filtered culture supernatant was separated on a 12.5% polyacrylamide gel under reducing or non-reducing conditions and transferred electrophoretically to polyvinylidene difluoride membranes (Millipore). The blots were blocked with 5% (w/v) non-fat dry milk (NFDM) in PBS containing 0.1% (v/v) Tween 20 (PBST) for 1 h and subsequently incubated overnight at 4°C with an anti-JHP0290 or anti- alkyl hydroperoxide reductase (AhpC) or anti-Urease α (bC-14) (Santa Cruz Biotechnology, SC-22445) antibody in blocking buffer. Protein A affinity-purified polyclonal antibody against JHP0290 and AhpC was generated by EZbiolab (USA). After washing with PBST, the membranes were incubated with a secondary antibody conjugated to the Odyssey IR-dye (Li-COR) for 1 h. The membranes were visualized using an Odyssey IR scanner (Li-COR). The band intensities of the immunoblots were quantified using the ImageJ software (NIH, Bethesda, MA, USA).