Fusion solo 7s

Plasmid pACYC-ipaBCDA was constructed by inserting T3SS effector genes (identical to nucleotide sequence 79825–91466; Genbank/EBI Data Bank Accession number CP000039.1) into the BamHI site of pACYC177 in a direction opposite to that of the tet promoter [26 (link)]. The bacterial strains used are listed in S1 Table. Strains MF1632 and MS2834 were constructed as previously described [27 (link)] using the primers listed in S2 Table.
For immunization or challenge, bacterial cells were grown at 37°C in LB Lenox medium as described previously [27 (link)], concentrated by centrifugation at 3,000 × g for 5 min at 4°C, and resuspended in PBS. Immunoblotting was performed as described previously [27 (link)]. To examine protein expression at 37°C, three independent cultures of 2457T and MF4835 were harvested at the same growth phase (OD₆₀₀ = 0.8), blotted onto the same membrane, and levels of IpaB and InvE were measured using a chemical luminescence-based imaging system (Fusion Solo 7S; VILBER Inc.). Values were calculated relative to those of 2457T (± the standard deviation).

The cells were gently scraped from the culture plates, resuspended in 1000 μL of Mammalian Protein Extraction Reagent buffer (Thermo Fisher Scientific), and shaken for 5 minutes. The samples were then centrifuged at 14,000g for 10 minutes. The supernatants were collected, and the protein concentration was calculated using a Qubit 2.0 fluorometer (Thermo Fisher Scientific). Protein extracts (30 μg per lane) were prepared, run on a 4%–20% Mini-PROTEAN TGX gel (Bio-Rad), and transferred to a 0.45 μm PVDF membrane. The membranes were blocked for 1 hour at room temperature using Blocking One (nacalai tesque), followed by incubation overnight at 4°C with the primary antibodies presented in Supplemental Table 1. Two secondary antibodies — anti-mouse IgG, HRP-linked whole Ab sheep (GE Healthcare, now Cytiva, NA931-1ML), and anti-rabbit IgG, HRP-linked antibody (Cell Signaling Technology, 7074S) — were used at a dilution of 1:5000, and the membranes were developed using ImmunoStar LD (Wako) and imaged using the FUSION Solo 7S (Vilber-Lourmat).